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THE AMINO ACID CONTENT AND NUTRITIVE VALUE 
OF THE PROTEINS OF COTTONSEED MEAL 



BY 



WILLIAM BARBOUR NEVENS 

B.S. University of Wisconsin, 1914 



THESIS 



Submitted in Partial Fulfillment of the Requirements for the 

Degree of 



DOCTOR OF PHILOSOPHY 



IN ANIMAL HUSBANDRY 



IN 

THE GRADUATE SCHOOL 

OF THE 

UNIVERSITY OF ILLINOIS 
1921 



THE AMINO ACID CONTENT AND NUTRITIVE VALUE 
OF THE PROTEINS OF COTTONSEED MEAL 



BY 



WILLIAM BARBOUR NEVENS 

B.S. University of Wisconsin, 1914 



THESIS 



Submitted in Partial Fulfillment of the Requirements for the 

Degree of 

DOCTOR OF PHILOSOPHY 

IN ANIMAL HUSBANDRY 

IN 

THE GRADUATE SCHOOL 
OF THE 

UNIVERSITY OF ILLINOIS 
1921 



^A' 



LIBRARY OF CONGRESS 

RECEIVED 

MAY 261922 

f)OCUM£NTvi D?VI3!Oi 






■■ij 



TABLE OF CONTENTS 

I. Amino Acid Content 

PAGE 

1. Introduction 375 

2. Methods Employed 377 

3. Discussion of Results 379 

4. Summary 398 

5. References i 399 

II. Nutritive Value 

1. Review of Previous Work 552 

2. Methods Employed 552 

3. Discussion of Results 573 

4. Summary 582 

5. References 588 



Digitized by the Internet Archive 
in 2011 with funding from 
The Library of Congress 



http://www.archive.org/details/aminoacidcontentOOneve 



Reprinted from Journal of Dairy Science 
Vol. IV, No. 5, September, 1921 



THE PROTEINS OF COTTONSEED MEAL 1 
I. AMINO ACID CONTENT 

W. B. NEVENS 
From the Department of Animal Husbandry, University of Illinois, Urbana 

I. INTRODUCTION 

One of the earliest references to the proteins of cottonseed 
meal was made by Ritthausen (1), who separated the pro- 
teins in the form of spheroids. Osborne and Voorhees (2) 
isolated a protein from cottonseed meal which had the nature of 
a globulin, being soluble in salt solutions, and comprised 42.3 
per cent of the total nitrogen of the meal. Another protein 
(or proteins) was found to be insoluble in salt solutions but 
soluble in 0.2 per cent potash solution and amounted to 44.3 
per cent of the total nitrogen of the meal. Two per cent of the 
total nitrogen was present in the form of water soluble proteose. 

TABLE 1 
Percentage of nitrogen in the different groups in various proteins 



Globulin, cottonseed 

Globulin, wheat 

Zein, maize 

Hordein, barley 



Nab 

AMMO- 
NIA 



1.92 
1.42 
2.97 
4.01 



BASIC 

N 



5.71 
6.83 
0.49 
0.77 



NON- 
BASIC 

N 



11.01 

9.82 

12.51 

12.04 



NlN 

MgO 

PRECIPI- 
TATION 



0.28 
0.16 
0.23 



TOTAL 

N 



18.64 
18.39 
16.13 
17.21 



The distribution of nitrogen in various protein bodies was 
studied exhaustively by Osborne and Harris (3), who employed 
the modified Hausmann method (4). The following values are 
typical of their results. 

1 The results presented in this paper formed part of a thesis submitted to the 
Graduate School of the University of Illinois in partial fulfillment of the require- 
ments for the degree of Doctor of Philosophy in Animal Husbandry. 

375 



JOURNAL OF DAIRY SCIENCE, VOL. IV, NO. 5 



376 - W. B. NEVENS 

These investigators state that "This wide variation in the 
proportion of basic decomposition products of the various pro- 
teins .... raises important questions regarding their food 
value." Osborne (5) further found that the basic nitrogen of 
the globulin of cottonseed, as determined by precipitation with 
phosphotungstic acid, consisted of 3.46 per cent histidine, 13.51 
per cent arginine and 2.06 per cent lysine. 

The content of the mono-amino acids of the "edestin" of 
cottonseed meal was determined by Abderhalden and Rostoski 
(6) by the use of the Fischer ester method (7), and is as follows, 
calculated for dry, ash free edestin of cottonseed: 

per cent 

Glycocoll . 1.2 

Alanin 4.5 

Amino valerianic acid Present 

a-proline 2.3 

Leucin 15.5 

Glutamic acid 17.2 

Aspartic acid 2.9 

Phenyl alanin 3.9 

Serin 0.4 

Tyrosin 2.3 

Tryptophane Present 

The quantitative determination of the amino acids of feeding- 
stuffs by means of the Van Slyke method (8) was undertaken 
by Grindley and his co-workers (9), and a little later by Nollau 
(10). In Nollau 's procedure, samples of the finely ground feeds 
were hydrolyzed with 20 per cent hydrochloric acid until the 
content of amino acid, as determined by the Van Slyke method, 
became constant. The material insoluble in hydrochloric acid 
was filtered off, the clear extract concentrated under diminished 
pressure and made up to a certain volume. The total nitrogen 
content of this extract was used as a basis for calculating the 
final results. In a report of the subsequent work of Grindley 
and co-workers (11), it is claimed that since Nollau filtered off 
the solid residue after hydrolysis of the feedingstuff and before 
making his total nitrogen determinations upon which the final 
calculations were based, that his results are not accurate since 
a part of the total nitrogen was undoubtedly discarded in the 
solid residue. 



THE PROTEINS OF COTTONSEED MEAL 377 

The heats of combustion of several vegetable proteins were 
carefully determined by Benedict and Osborne (12). The 
globulin of cottonseed was found to yield 5598 calories per 
gram, compared to 5358 calories per gram for the globulin of 
wheat and 5916 calories per gram in the case of the hordein of 
barley. In commenting upon their determinations, the inves- 
tigators state that " many irregularities. . . . appear, which 
are doubtless due to the different proportion of the various amino 
acids which constitute the molecules of the different proteins." 

It is evident from the foregoing discussion that our knowledge 
of the composition of the proteins of cottonseed meal is very 
incomplete. The globulin is the only protein of cottonseed 
which has been isolated in pure form and whose composition 
has been determined. The globulin, however, according to 
Osborne and Voorhees, contains only 42.3 per cent of the total 
nitrogen of the cottonseed. The character, identity and chemical 
composition of the remaining proteins are practically unknown, 
and it is evident from the data given above that our knowledge 
of the distribution of the nitrogen in the proteins of cottonseed 
meal is very meager indeed. The investigation of the distribu- 
tion of the nitrogen in the proteins of cottonseed meal therefore 
constituted the object of this study. 

II. METHODS EMPLOYED IN CHEMICAL ANALYSIS 2 

The method of analysis employed consisted of two main 
procedures. The first consisted of a series of extractions whereby 
the nonprotein together with a very small amount of protein was 
first removed, and following this, the proteins were extracted 
from the residual matter of the sample which consisted mostly of 
fiber. The second main procedure embraced the hydrolysis of 
the extracted proteins, and the analysis of the resulting solution 

2 The method of procedure here outlined is one which has been developed and 
perfected in this laboratory by Dr. H. S. Grindley, Mr. T. S. Hamilton and asso- 
ciates (9, 11, 33, 36 and unpublished manuscripts). The method of extraction 
preliminary to hydrolysis of the proteins has been developed entirely in this 
laboratory, while the actual determination of the nitrogen in the different groups 
follows closely the method of Van Slyke, but includes modifications perfected 
in this laboratory. 



378 W. B. NEVENS 

for certain amino acid and other groups according to the general 
method of Van Slyke (8). 

The sample of cottonseed meal was prepared from good 
quality commercial meal, finely ground and passed through a 40- 
mesh sieve. Each sample taken for analysis weighed 15 grams 
and contained 1.0194 grams of nitrogen, or about 6 grams of 
protein. 

In the first three extractions, which were carried out con- 
secutively, cold anhydrous ether, cold absolute alcohol and 
cold 1.0 per cent trichloracetic acid were used. The samples of 
feeding stuff were placed in 500 cc. centrifuge bottles and 100 
to 200 cc. of the reagents added. The bottles were placed on 
a shaking machine which rolled them back and forth continually. 
Usually two extractions were made each twenty-four hours, one 
extraction period being seven to eight hours and the other 
15 to 16 hours in length. At the end of the extraction period, 
the sides of the bottles were washed down with the reagent, the 
bottles centrifuged and the clear supernatant liquid decanted. 
Usually six or seven extractions with each reagent were necessary. 

The ether and alcohol extracts were filtered and any residues 
returned to the centrifuge bottles. After slight acidification 
with sulphuric acid, the ether and alcohol were evaporated and 
recovered and total nitrogen determinations made on the residue. 
The small amount of protein removed in the trichloracetic acid 
extracts was recovered by precipitation with colloidal ferric 
hydroxide (containing 5 per cent Fe 2 3 ) in boiling solution. 
The precipitate was transferred to a digestion flask with 20 per 
cent hydrochloric acid, and total nitrogen determined in the 
filtrate. 

The bulk of the proteins was removed from the residue re- 
maining after extraction with 1.0 per cent trichloracetic acid 
by extraction, first, with dilute sodium hydroxide solution, then 
with 20 per cent hydrochloric acid followed by treatment with 
5 per cent sodium hydroxide solution. The dilute sodium 
hydroxide solution used during the shorter period was a 0.2 per 
cent solution and that during the longer period a 0.1 per cent 
solution. These extracts were neutralized, acidified with hydro- 



THE PROTEINS OF COTTONSEED MEAL 379 

chloric acid and concentrated in vacuo to a small volume. An 
equal volume of concentrated hydrochloric acid was then added. 
The residues remaining after treatment with dilute sodium 
hydroxide solution were boiled for three minutes with 20 per cent 
hydrochloric acid. After cooling, the solution was filtered 
off, the residue washed and the procedure repeated once. The 
washings were evaporated to a small volume, an equal volume 
of concentrated hydrochloric acid added and the washings then 
combined with the main hydrochloric acid extract. The residues 
insoluble in hydrochloric acid were treated three times with 5 
per cent sodium hydroxide solution using centrifuge bottles as 
in former extractions. After washing the residues nearly free 
from alkali, they were submitted to Kjeldahl analysis. The 
extracts and washings were acidified with hydrochloric acid, 
concentrated in vacuo and transferred to digestion flasks with 
an equal volume of concentrated hydrochloric acid. 

The proteins precipitated by colloidal iron and the proteins 
removed by extraction with dilute sodium hydroxide, 20 per 
cent hydrochloric acid and 5 per cent sodium hydroxide were 
completely hydrolyzed by boiling for fifteen hours upon a 
combined electric plate and sand bath under reflux condensers. 
The resulting solutions were combined and analysis for the 
chemical groups characteristic of certain amino acids executed 
essentially as directed by Van Slyke (8), but with the use of 
minor improvements perfected in this laboratory. 

III. DISCUSSION OF RESULTS 

The results obtained by application of the method of chemical 
analysis outlined in the preceding section to eight portions of the 
same original sample of cottonseed meal are shown in the accom- 
panying tables 2 and 3. Table 2 shows the values expressed in 
percentage of the total nitrogen present in the sample of feeding 
stuff when it was taken for analysis, while table 3 shows the same 
values expressed in percentage of the feeding stuff itself. 

Two averages are included in the tables. The first is com- 
piled by taking the average of all values obtained by analysis of 
the entire eight samples. The second average is obtained by 



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384 W. B. NEVENS 

averaging the results secured in the analysis of the complete 
samples C2, C3, C6 and C7. It is believed that the latter 
average more nearly expresses the actual composition of the 
commercial cottonseed meal used, for the following reasons: 
(a) These samples, i.e., C2, C3, C6 and C7 show the best agreeing 
results throughout. The two parts of sample CI agree well in 
the amount of arginine nitrogen, but show a considerable differ- 
ence in the amounts of amino nitrogen, non-amino nitrogen and 
histidine nitrogen. The totals of the nonprotein nitrogen plus 
the protein nitrogen are considerably below the average of all the 
samples. In sample C4 the non-amino nitrogen is particularly 
low, this value being one of the principal factors contributing 
to the noticeably low nonprotein plus protein of this sample. 
Sample C5 is omitted from the average partly on account of the 
non-agreement of its arginine nitrogen and histidine nitrogen 
values. Of the latter values, one is 3 per cent above the average 
of all samples, (b) The second average, i.e., of samples C2, 
C3, C6 and C7, includes values which are most free from obvious 
errors. In making the determinations in the case of sample 
C4, two determinations were lost, and in the case of sample C5, 
one determination was lost. While the results obtained for sam- 
ple C8 agree fairly well throughout with themselves and with the 
average, the results are consistently high, and it is excluded 
from this average on the grounds of the totals obtained, which 
are obviously too high. 

Nonprotein nitrogen. The first section of tables 2 and 3 shows 
the amount of nitrogen removed in the preliminary extractions 
with absolute ether, absolute alcohol and trichloracetic acid. 
While the absolute ether in the cold is used primarily to remove 
the lipins, such as the oils, waxes, etc., it also dissolves various 
amounts of other substances, such as coloring matters, and at the 
same time a small amount of nitrogen. The thorough extrac- 
tion with absolute alcohol following the treatment with absolute 
ether presumably completes the extraction initiated by ether. 
The alcohol removes somewhat more nitrogen than the extrac- 
tion with ether. The bulk of the nonprotein nitrogen accounted 
for, about 89 per cent of the total, remains, however, in the tri- 



THE PROTEINS OF COTTONSEED MEAL 385 

chloracetic acid extracts after precipitation of the proteins by 
colloidal ferric hydrate and the removal of the precipitate by 
filtration. That the nitrogen determined in these latter extracts 
is not protein nitrogen is apparent from the work of Van Slyke, 
Vinograd, Vilchur and Losee (13), Hill (14), Wolff (15), and 
others. 

It seems from the study of the character of the nonprotein 
nitrogenous constituents of feedingstuffs by Grindley and 
Eckstein (16) that the forms of nitrogen represented in this 
classification consist principally of those forms naturally re- 
sulting from the cleavage of the proteins upon hydrolysis and 
therefore could not interfere in the determination of the charac- 
teristic chemical groups of the proteins were they not removed 
in the preliminary extractions. Neidig and Snyder (17), who 
recently determined the proportion of nitrogen in the form of 
ammonia in the ether extracts and alcohol extracts of different 
kinds of silage, found that from 28.1 per cent to 100 per cent of 
the ether extract nitrogen consists of ammonia nitrogen, while 
from 14.2 per cent to 23.4 per cent of the alcohol extract nitrogen 
is yielded as ammonia nitrogen. This indicates that only a 
part of the nitrogen soluble in ether and alcohol would appear 
in the ammonia fraction were it not removed previous to hydrol- 
ysis. Therefore, the removal of the nonprotein nitrogen at 
this point avoids possible complications in the further prose- 
cution of the analytical procedure, and it is believed that the 
accuracy of the further determinations has been increased over 
that of previous methods by the removal of the nonprotein 
nitrogen before hydrolysis of the proteins. The total amount of 
the nonprotein nitrogen present in cottonseed meal found by 
the method used amounted to 6.106 per cent of the total nitrogen 
contained in the feedingstuff. 

Results of the Van Slyke analysis. It is a matter of common 
knowledge that one of the important sources of loss in the analy- 
sis of proteins by methods involving the employment of acid 
hydrolysis is the formation of an insoluble black substance called 
humin. The term melanin is also applied to this substance, 
on account of its supposed relationship or similarity to the 



386 W. B. NEVENS 

naturally occurring body pigments. The amount of humin formed 
in acid hydrolysis of the proteins is greatly increased by the 
presence of carbohydrates, as shown by Gortner and associates 
(18, 19), Hart and Sure (20), and Osborne, Van Slyke, Leaven- 
worth and Vinograd (21). A part of the humin formed, how- 
ever, remains in solution in the hydrochloric acid, and is 
termed soluble humin. 

In these experiments the soluble humin which is adsorbed by 
the lime used in neutralizing the hydrolysate when determining 
ammonia nitrogen, carried with it a larger amount of nitrogen 
than the insoluble humin. The sum of the insoluble plus the 
soluble humin nitrogen found amounts to 6.589 per cent, which 
constitutes no inconsiderable error, since at present it is im- 
possible to determine the character of the nitrogen discarded in 
this form. 

It may be noted by referring to table 1 that the amount of 
soluble humin nitrogen in the first four samples is considerably 
greater than in the succeeding four. Possibly this is due to a 
slight variation in the analytical procedure. In the case of 
samples CI to C4, inclusive, it was necessary to add 75 to 90 
cc. of calcium hydroxide in order to neutralize the hydrochloric 
acid before distillation of ammonia. About 20 cc. in excess were 
then added. With the next four samples evaporation of the 
acid hydrolysate in vacuo was continued longer in order to 
drive off a greater proportion of the hydrochloric acid. In conse- 
quence, only 30 to 40 cc. of calcium hydroxide were necessary 
to effect neutralization, and in these cases only a small excess, 
about 10 cc, of calcium hydroxide was added. The hypothesis 
is put forward that the presence of a large excess of calcium 
hydroxide during the distillation of ammonia may result in the 
adsorption of some amino acid nitrogen which is incompletely 
removed in the subsequent washing of the sticky mass. 

When it is recalled that the proteins of cottonseed meal form 
approximately 43 per cent of the feedingstuff, the amount of 
nitrogen in the humin resulting from the hydrolysis of these 
proteins, as determined in these experiments, is not excessive 
when compared to the amounts obtained in the hydrolysis of 



THE PROTEINS OF COTTONSEED MEAL 387 

pure proteins by Van Slyke (8) , some of whose results are shown 
in the accompanying table. 

The amount of nitrogen recovered as ammonia was quite 
constant in all the samples. Little can be said in regard to the 
significance of this fraction, aside from the fact that the propor- 
tion of the total nitrogen of cottonseed meal which appears as 
ammonia is quite in harmony with that of other feedingstuffs. 

A particularly characteristic feature of the amino acid con- 
tent of cottonseed meal is the remarkably high content of argi- 
nine. This is much higher than that found in any other feeding 
stuff so far examined, with the exception of peanuts, although it 
is not so high as that found in some other vegetable proteins. 
Van Slyke (8) found 27.05 per cent arginine nitrogen in edestin 

TABLE 4 
Amounts of humin nitrogen in pure proteins expressed in percentage of the total 

nitrogen of the protein 



PROTEIN AND DESCRIPTION 



Gliadin from wheat 

Edestin 

Fibrin (Merck's) 

Oxyhemoglobin ("pure, crystalized") . 
Dog's hair 



HUMIN NITROGEN 



per cent 

0.86 
1.83 
3.43 
3.60 
7.35 



while Nollau reported that hemp seed, peanuts, black walnuts, 
and hickory nuts have an arginine nitrogen content of more 
than 20 per cent. 

In the Van Slyke procedure the only amino acids deter- 
mined by direct analysis are arginine and cystine. Just how 
much importance may be attached to the results obtained for the 
latter is questionable, even though the values found in the 
different samples do not vary widely. These values, however, 
probably fall short of the true value, due to losses in the deter- 
mination of cystine. Van Slyke (8) has shown that boiling 
cystine for sixteen hours with hydrochloric acid resulted in the 
conversion of one-half of its nitrogen into forms not precipitable 
by phosphotungstic acid. Since in the analytical procedure de- 
scribed above, hydrolysis of the proteins was carried out by 



388 W. B. NEVENS 

boiling them with 20 per cent hydrochloric acid for fifteen hours, 
it is probable that much of the cystine was destroyed during 
that reaction. If it is assumed that the correct value for cys- 
tine should be double that actually obtained, then the total 
nitrogen of the bases would amount to 31.75 per cent of the 
total nitrogen of the feedingstuff. 

The values given for histidine and lysine are somewhat vari- 
able among the different samples. These variations are likely 
due in large measure to the indirect method used in their deter- 
mination, since slight errors in any or all of the three direct 
determinations of arginine nitrogen, cystine nitrogen and the 
total nitrogen of the bases are doubtless all reflected at these 
points. 

The content of mono-amino acid nitrogen of cottonseed meal 
is considerably less than that found in other feedingstuffs, 
possibly due to the greater proportion of the total nitrogen 
which is formed by the basic amino acids. One of the interest- 
ing features of the results of the Van Slyke analysis of the pro- 
teins of cottonseed meal is brought out in the summation of the 
ammonia nitrogen, the nitrogen of the bases, mono-amino 
acid nitrogen, and non-amino acid nitrogen, the four groups 
which represent the total content of strictly amino acid nitro- 
gen as determined by this method. The sum of these is 83.132 
per cent. While this sum is not so great as that in the case of 
some other feedingstuffs or of animal proteins, as determined by 
previous investigators employing the Van Slyke method of 
analysis, it is a much larger amount than it was possible to 
secure in most cases from comparable sources by the methods of 
isolation and purification employed by the earlier investigators. 
Thus the tabulations of Lusk (22), combining the results of 
Osborne and associates in this field, show the maximum amino 
acid content of zein of maize, to be 88.87 per cent and that of 
gliadin of wheat as 85.68 per cent, but in the majority of cases 
the sum of the nitrogen content of the amino acids actually 
isolated from vegetable proteins ranges from 50 to 65 per cent 
of the total nitrogen of the protein. 



THE PROTEINS OF COTTONSEED MEAL 389 

Uncharacterized nitrogen lost in analysis. In the various 
steps of the analytical procedure small amounts of nitrogen of 
unknown character are included in residues and solutions which 
are discarded. In general, these have been disregarded by 
workers in other laboratories, especially those losses occurring 
at points indicated in table 2 by the last four of the subheadings 
included under the heading " Uncharacterized nitrogen lost in 
analysis," but in this laboratory the nitrogen discarded at each 
of these steps has been determined. Under the above mentioned 
headings, it is shown that, on the average, only 0.492 per cent 
of the total nitrogen remains in the residues insoluble in strong 
alkali, or in other words 99.508 per cent of the total nitrogen 
of the f eedingstuff is extracted as a result of the method employed, 
and that in individual cases as much as 99.78 per cent of the 
total nitrogen present was removed. As previous workers 
failed to isolate the proteins from the feedingstuff before hy- 
drolysis, it is not established that part of the insoluble residue 
discarded in their methods did not include some nitrogen in the 
form of non-hydrolyzed protein although this does not seem 
highly probable. It is believed, however, that the nearly 
complete extraction of the proteins before hydrolysis lends to the 
accuracy of the method by facilitating hydrolysis and in reducing 
the amount of humin. 

The largest item of loss occurs in the residue which remains 
after dissolving the precipitate of the bases in the amyl alcohol- 
ether mixture. This, presumably, is soluble humin which has 
not been adsorbed by the lime in the determination of ammonia, 
and fouls the solution at this point. This difficulty was also 
encountered by Menaul (23), who employed a preliminary 
precipitation with phosphotungstic acid in boiling solution for 
the separation of the humin and ammonia before the precipita- 
tion of the bases. In the present investigation, very little of the 
soluble humin appeared when the bases were precipitated, in 
most cases the precipitates being free from black particles. 
Washing with alternate portions of amyl alcohol-ether and 
water and then taking up the residue and washing thoroughly 
with water seemed to have little effect in reducing this source 



390 W. B. NEVENS 

of loss. A considerable portion of the nitrogen lost is soluble 
in the amyl alcohol-ether mixture, while smaller losses occur 
in the residues resulting from concentration of the solutions of 
the bases and filtered from the bases. Presumably, the second, 
third and fourth items of loss include some nitrogen which 
should be credited to the bases, but the character of this nitrogen 
was not determined. If these losses can be reduced, the total 
nitrogen of the bases of cottonseed meal may be found to be 
somewhat greater than the amount here reported. 

Total nitrogen accounted for. Summation of the nitrogen 
found in the various fractions of the protein molecule together 
with that in the unavoidable losses in the procedure gives totals 
which average 98.75 per cent. While the use of the Van Slyke 
method of analysis has enabled others to account for as great 
a proportion of the nitrogen of feedingstuffs, it is doubtful, 
for reasons pointed out below, if their results give as accurate a 
picture of the distribution of nitrogen in feedingstuffs as is 
obtained by the procedure employed in the present investiga- 
tion. 

Physiological significance of the basic amino acids. Our knowl- 
edge of the physiological role of arginine and histidine has been 
enhanced by the studies of Ackroyd and Hopkins (24). Em- 
ploying rations in which the nitrogen was provided in the form 
of hydrolyzed casein from which these two amino acids had 
been removed by precipitation according to the method of 
Kossel and Kutcher, it was found that rats receiving these 
rations declined rapidly in weight, but that when either amino 
acid was returned to the ration, loss in weight was prevented and 
some growth ensued. The investigators suggest that possibly 
either of these amino acids may be converted into the other 
by the animal body. It was further observed that when argi- 
nine and histidine are removed from the ration, the excretion of 
allantoin, which is the main end product of purine metabolism 
in the rat, was lowered. Subsequent experiments proved that 
the falling off of allantoin excretion was not due to lowered me- 
tabolism. From these observations and from the fact that the 
arginine, histidine and guanine molecules have similar structural 



THE PROTEINS OF COTTONSEED MEAL 391 

relationships, it was concluded that possibly one of the functions of 
arginine and histidine is to furnish the raw material for the purine 
metabolism of the animal organism. 

The above conclusion regarding the importance of arginine in 
purine metabolism is given added weight by the findings of 
Myer and Fine (25) regarding the creatine content of muscle. 
Differences of as much as 2.5 per cent in the creatine content of 
muscle were noted as a result of feeding rations high and low in 
arginine. 

That cystine plays an important part in nutrition has been 
brought out by several investigators, among them Osborne and 
Mendel (26). The latter obtained adequate growth by the 
addition of cystine to rations containing 9 per cent of casein, on 
which growth had been limited. Geiling (27), working in this 
laboratory, concluded that cystine seems to be necessary for the 
maintenance of adult mice. The importance of cystine to the 
animal organism is admirably set forth by Matthews (28). 

In the intermediary metabolism of the body, that is, the metabolism 
of the tissue, sulphur probably plays a very important role. This is 
shown not only by the fact that it is absolutely necessary for the 
continued existence of the body, as necessary as nitrogen or any of the 
other elements, but also by the fact that it is one of the most labile 
elements of the protein molecule. No other element is split off from 
the proteins with greater ease than this. It is, indeed, the labile 
element par excellence. Moreover, cysteine, which is one of the amino 
acids, readily oxidizes itself. It is a reducing body. It oxidizes 
spontaneously and there are many points in its oxidation which strongly 
resemble the process of respiration. Thus the most favorable concen- 
tration of hydrogen ions for the oxidation of cysteine is the same as 
that in protoplasm; both cysteine and protoplasm are poisoned by 
many of the same substances, such as the nitriles, the cyanides, acids, 
and the heavy metals; their oxidations are catalyzed or hastened in 
the same manner by iron, arsenic and some other agents. For these 
reasons it has been suggested by Hefter and the author that there is 
more than a superficial connection between the oxidation of cysteine 
and the respiration of the cell. 



392 W. B. NEVENS 

The necessity of lysine for growth has been conclusively dem- 
onstrated by Osborne and Mendel (29). When gliadin of 
wheat, which contains only a minute amount of lysine, formed 
the sole source of protein in the rations of rats, the live weight of 
the animals was maintained over long periods, but normal 
growth could not be secured. When lysine was added to the 
rations, normal growth occurred. In other investigations (30), 
in which zein of maize was used as the source of the protein, 
it was found that a rat could be maintained at an almost constant 
weight of 50 grams for a period of one hundred and eighty-two 
days when tryptophane was added to the extent of 3 per cent 
of the zein. The further addition of lysine induced normal 
growth. Further study (31) of the necessity of lysine in the 
ration convinced these investigators that about 2 per cent of 
the protein of the ration must consist of lysine in order to pro- 
mote normal growth in the rat. Osborne and Mendel (32) also 
demonstrated the necessity of lysine for the growth of chickens. 

In view of the essential role which the basic amino acids play 
in nutrition as brought out above, it is reasonable to assume from 
a survey of the analytical results of cottonseed meal secured in 
this investigation that the proteins of this feedingstuff have a 
high nutritive value. The combined arginine and histidine 
content of cottonseed meal is greater than that of any other 
feedingstuff so far analyzed with the exception of the peanut. 
This feature alone is of great importance in view of the fact 
that arginine and histidine seem to be interchangeable in nutri- 
tion. While the lysine content cannot be said to be exceptional 
in any particular it seems apparent from the above discussion, 
that the combined proteins of cottonseed meal contain sufficient 
amounts of both cystine and lysine to render them adequate for 
nutrition. 

Comparison with previous analyses of cottonseed meal. As 
shown in the introduction, there are but few determinations of 
the chemical composition of the proteins of cottonseed meal. 
The earliest studies were made upon one of the isolated pro- 
teins, the globulin or "edestin" of cottonseed, the results of 
which can not well be compared to analyses of the combined 



THE PROTEINS OF COTTONSEED MEAL 



393 



proteins, since, as previously mentioned, the globulin contains 
but 42.3 per cent of the total nitrogen of cottonseed meal. In 
the accompanying table 5, the values secured by two previous 
investigators who made analyses of the combined proteins of 
cottonseed meal are brought together for comparison with those 
obtained in this investigation. 

It is evident from the results presented that there is a general 
agreement between the three sets of values, but that there are 
considerable differences in several important particulars. As 
pointed out in the introduction, Nollau (10) calculated his re- 
sults upon the total nitrogen content of the hydrolyzed solution 
after filtering off the solid residue insoluble in hydrochloric 

TABLE 5 

Distribution of nitrogen in cottonseed meal as determined by different investigators 

(Results expressed in percentage of the total nitrogen of the feeding stuff) 



INVESTIGATOR 


HUMIN 
N 


AMMO- 
NIA N 


ARGI- 

NINE 
N 


CYSTINE 

N 


HISTI- 
DINE N 


LY- 
SINE 

N 


AMINO 

' N 

IN FIL- 
TRATE 
FROM 

BASES 


NON- 
AMINO 

N 

IN FIL- 
TRATE 
FROM 

BASES 


TOTAL 

N 

ACCOUNT- 
ED FOR 


Nollau 


6.27 

7.78 
6.58 


14.06 

10.45 

9.49 


12.77 
19. '52 

18.74 


2.74 
0.65 
0.91 


7.57 
5.47 
7.40 


1.94 
4.78 
3.81 


45.02 
42.82 
40.12 


7.49 
5.43 
2.68 


97.48 


Grindley 


96.90 


Nevens 


98. 75 1 







1 Includes 9.03 per cent N removed in preliminary extractions plus unchar- 
acterized nitrogen lost in method of analysis. 

acid. This means that all of his calculations are too high, 
since a part of the nitrogen of the sample was undoubtedly dis- 
carded in the solid residue. The value of 6.27 per cent humin 
nitrogen reported by Nollau must, therefore, represent the 
soluble humin nitrogen, which is nearly as large a value as 
that obtained by the writer for the sum of the insoluble humin 
nitrogen plus the soluble humin nitrogen. The amount of 
soluble humin .nitrogen found by the writer was but 3.89 per 
cent. Compared to the total humin nitrogen found by Grindley, 
et al. (9), the amount of total humin nitrogen as determined 
by the writer was 1.19 per cent less. The reduction of the 
humin nitrogen has no doubt been an important contributing 



394 W. B. NEVENS 

factor in the present investigation in securing somewhat higher 
values of the basic amino aids. In view of the known effects 
of acid hydrolysis of the proteins in the presence of carbohy- 
drates, as already pointed out, it is reasonable to assume that 
the smaller amount of nitrogen discarded in the form of humin 
in these experiments than in those of Grindley may be attributed 
to the more complete separation of the proteins from the carbo- 
hydrates before hydrolysis. 

The method of analysis of the proteins after hydrolysis by 
hydrochloric acid, as employed by Grindley et al., was similar 
to that employed by the writer, the main point of difference 
between the complete procedures being in the omission by the 
former workers of the extractions previous to hydrolysis. At 
just what point the 6.106 per cent of nonprotein nitrogen re- 
moved by the writer in the preliminary extractions might 
appear were it not so removed, is not clear. However, the sum 
of the ammonia nitrogen, amino nitrogen and non-amino nitro- 
gen in the nitrate from the bases, obtained by Grindley et al, is 
6.414 per cent greater than the sum of the corresponding values 
obtained by the writer, so it is possible that these three forms of 
nitrogen as reported by the former comprise some nitrogen not 
derived from the proteins as such. 

It is evident from the table that the nitrogen of the bases as 
found by Grindley and his coworkers are in much closer agree- 
ment with those obtained by the writer than those reported by 
Nollau. The latter 's figures for arginine are obviously too 
low, while his cystine values are more than four times as great 
as those of Grindley et al. and three times as great as those of 
the Writer. Accordingly, the lysine nitrogen values as calculated 
by Nollau are correspondingly too low. The values for the total 
nitrogen of the bases as found by the three investigators in the 
order given in the table are as follows: 25.02 per cent, 30.42 
per cent and 30.84 per cent respectively, the last being nearly 
0.5 per cent higher than previous determinations. 

The total nitrogen accounted for in the three reports is like- 
wise shown to be 97.86 per cent, 96.90 per cent and 98.75 per cent. 
The greater amount in the last case is evidently due in part 



THE PROTEINS OF COTTONSEED MEAL 395 

at least, to the inclusion of the determinations of the uncharac- 
terized nitrogen lost at points were unavoidable losses occur in 
the method of analysis. These losses were not determined by 
the first two investigators. 

Comparison of the distribution of nitrogen in cottonseed meal 
with that in other feedingstuffs. A comparison of the results of 
analysis of the proteins of cottonseed meal, as discussed above, 
with those obtained by Hamilton, Grindley and Nevens (33) 
for alfalfa hay, oats and corn is of value in studying the relative 
nutritive value of the proteins of these feedingstuffs, as well as 
the applicability of the general method of analysis to feeding- 
stuffs which vary widely in composition. In the analysis of 
oats and corn an additional preliminary extraction, which 
involves the use of hot trichloracetic acid, is employed to remove 
the starch. This extraction is not necessary in the case of 
cottonseed meal and alfalfa hay on account of the absence of 
starch in the former, as stated by Withers and Fraps (34), 
and the relatively small amount of starch in the latter. 

The first point of interest in contrasting these feedingstuffs, as 
may be noted by reference to table 6, is their content of non- 
protein nitrogen. Oats contain more than twice as much non- 
protein nitrogen as cottonseed meal, while alfalfa hay contains 
more than three times as much. Hart and Bentley (35) found 
that 23.5 per cent of the nitrogen of alfalfa hay is present in a 
water soluble form, while Grindley and Eckstein (16) found a 
value of 28.4 per cent for the same feedingstuff. 

The amount of total humin is greatest in the case of alfalfa, 
a natural result, since the proteins are more difficultly extracted 
from those feedingstuffs containing large amounts of crude fiber. 
The amount of humin in the case of corn is very small indeed, 
considering the high percentage of carbohydrates in this cereal, 
and compares very favorably with the amounts of humin re- 
sulting from the hydrolysis of pure proteins as shown in table 3. 
Cottonseed meal occupies a medium position in respect to the 
proportion of humin nitrogen. 

The most striking difference between these four feeding- 
stuffs is in their basic amino nitrogen content. Cottonseed 



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396 



THE PROTEINS OF COTTONSEED MEAL 397 

meal, as already indicated, is exceptionally high in arginine nitro- 
gen, but it is also much higher in its total basic nitrogen con- 
tent than the other three feedingstuffs, the values for the four 
feedingstuffs being: alfalfa hay, 17.412 per cent; oats, 21.228 
per cent; corn, 17.529 per cent; and cottonseed meal, 30.846 per 
cent. The sum of the arginine nitrogen and histidine nitrogen is 
more than twice as great in the case of cottonseed meal as in 
the case of alfalfa hay and nearly twice as great as that of corn. 
From the considerations presented above regarding the biological 
significance of the basic amino acids, it would be logical to 
assume that these wide differences in the chemical composition 
of the proteins of different feedingstuffs indicate similar differ- 
ences in their nutritive value, though probably not in corre- 
sponding degree. This point is mentioned in another paper in 
connection with the discussion of the results of the feeding 
experiment conducted for the purpose of studying the nutri- 
tive value of the proteins of cottonseed meal. 

Alfalfa hay contains the smallest proportion of mono-amino 
acid nitrogen, possibly owing to its high content of nonprotein 
nitrogen while corn is exceptionally high in its content of 
both mono-amino and non-amino acid nitrogen. 

The largest amount of nitrogen lost in the method of analy- 
sis occurs in the case of alfalfa, which is accounted for largely 
in the nitrogen remaining in the residues after the preliminary 
extractions have been completed. The next largest amount 
is in the case of corn, where the bulk of the loss is due to un- 
adsorbed humin. The nitrogen is extracted very completely 
from both oats and corn. Cottonseed meal occupies a medium 
position with respect to the nitrogen lost in the analytical 
procedure. 

The total nitrogen accounted for in the case of the various 
feedingstuffs is a point worthy of special note. The total is 
least in the case of alfalfa and greatest with corn. Here again 
cottonseed meal occupies a medium position. In this rather long- 
method of analysis, which involves many extractions, concentra- 
tions, precipitations, nitrations and transfers, and which at some 
stages renders the proteins subject to putrefaction unless care 



398 W. B. NEVENS 

is taken, only 0.101 per cent of the total nitrogen originally 
present in the sample of corn was not accounted for, a very 
remarkable result indeed. 

An examination of the results of individual analyses of the 
four samples of alfalfa hay and six samples each of oats and corn 
which were averaged to obtain the values shown in table 6, 
brings out the fact that the analytical results in the case of each 
of these feedingstuffs show, on the whole, less variability than 
the values for the eight samples of cottonseed meal shown in 
table 2. At least two factors operated to effect the difference. 
The analyses of the first three feedingstuffs mentioned were 
conducted by persons experienced in the manipulation and 
execution of the Van Slyke analysis and the analyses used for 
the averages were selected from a number of analyses. The 
analyses of cottonseed meal were made by the writer who had 
had no previous experience in the conduct of the Van Slyke 
method, and the analyses presented in table 1 are the entire 
results of the work. These considerations are strong evi- 
dence that the method of analysis here described is of general 
application to feedingstuffs and may readily be carried out. 

Summary of the discussion of the results of the chemical analysis 
of the proteins. The accuracy of the determination of the amino 
acid content of the proteins of cottonseed meal has been in- 
creased over that of previous methods by the removal of the 
nonprotein nitrogen before proceeding with the hydrolysis of the 
proteins. 

The accuracy of the determination has been still further 
increased by the reduction of the humin substances formed as 
a result of the hydrolysis of the proteins. 

The amount of arginine nitrogen is much higher than that 
in most other feedingstuffs. The sum of the four basic amino 
acids is about 0.5 per cent higher than values previously found 
for cottonseed meal. 

The method of extraction employed was found to result in 
the removal of 99.5 per cent of the total nitrogen present in the 
feedingstuff. 



THE PROTEINS OF COTTONSEED MEAL 399 

The sum of the ammonia nitrogen and amino acid nitrogen 
fractions is 83.132 per cent of the total nitrogen, an amount 
comparable to the sum of the same fractions previously ob- 
tained from pure vegetable proteins. 

Of the total nitrogen originally present in the sample of cotton- 
seed meal, 98.75 per cent was accounted for by summation of 
the fractions obtained at different stages in the method of 
analysis, a proportion greater than any previously reported for 
the same feedingstuff. 

The complete method of analysis outlined in this paper is 
believed to be of general application to feedingstuffs and may 
readily be executed with successful results. 

The writer is greatly indebted to Dr. H. S. Grindley for many 
courtesies extended during the course of this investigation in 
addition to his constructive criticism and general supervision 
of the work. His thanks are also due Mr. T. S. Hamilton for 
advice regarding certain analytical procedures. 

REFERENCES 

(1) Ritthatjsen, H. : Jour. f. prak. Chem. (Neue Folge), 1881, xxiii, 481. 

(2) Osborne, T. B., and Voorhees, C. G. : Jour. Amer. Chem. Soc, 1894, xvi, 

778; Annual Rept. Conn. Agr. Exp. Sta., 1893, xvii, 211. 

(3) Osborne, T. B., and Harris, I. F. : Jour. Amer. Chem. Soc, 1903, xxv, 323. 

(4) Hatjsmann, W.: Zeit. physiol. Chem., 1899, xxvii, 95; ibid., 1900, xxix, 136. 

(5) Osborne, T. B.: The Vegetable Proteins. 1912, p. 59. 

(6) Abderhalden, E., and Rostoski, O. : Zeit. physiol. Chem., 1905, xliv, 265. 

(7) Fischer, E : Zeit. physiol. Chem., 1901, xxiii, 151. 

(8) Van Slyke, D. D.: Jour. Biol Chem., ix, 185; ibid., 1911, x, 15; ibid., 1912, 

xii, 275; ibid., 1915, xxii, 281. 

(9) Grindley, H. S., Joseph, W. E., and Slater, M. E. : Jour. Amer. Chem. 

Soc, 1915, xxxvii, 1778; Sc, 1915, xlii, 70. 

(10) Nollau, E. H. : Jour. Biol. Chem., 1915, xxi, 611. 

(11) Grindley, H. S., and Slater, M. E. : Jour. Amer. Chem. Soc, 1915, 

xxxvii, 2762; Proc Soc. An. Prod., 1917, p. 133. 

(12) Benedict, F. G., and Osborne, T. B.: Jour. Biol. Chem., 1907, iii, 119. 

(13) Van Slyke, D. D., Vinograd-Villchur, M., and Losee, J. R. : Jour. 

B ol. Chem., 1915, xxiii, 377. 

(14) Hill, R. L.: Jour. Biol. Chem., 1915, xx, 175.- 

(15) Wolff, C. G. L.: Jour. Physiol., 1915, xlix, 89. 



400 W. B. NEVENS 

(16) Grindley, H. S., and Eckstein, H. C. : Jour. Amer. Chem. Soc, 1916, 

xxxviii, 1425. 

(17) Neidig, R. E., and Snyder, R. S. : Jour. Amer. Chem. Soc, 1921, xliii, 951. 

(18) Gortner, R. A., and Blish, M. J.: Jour. Amer. Chem. Soc, 1915, xxxvii, 

1630; Jour. Biol. Chem., 1916, xxvi, 177. 

(19) Gortner, R. A., and Holm, G. E. : Jour. Amer. Chem. Soc, 1917, xxxix, 

2477 and 2736. 

(20) Hart, E. B., and Sure, B.: Jour. Biol. Chem., 1916, xxviii, 241. 

(21) Osborne, T. B., Van Slyke, D. D., Leavenworth, C. S., and Vinograd, 

M.: Jour. Biol. Chem., 1915, xxii, 259. 

(22) Lusk, G. : The Science of Nutrition. 3d Ed. 1919, p. 77. 

(23) Menattl, P.: Jour. Biol. Chem., 1921, xlvi, 351. 

(24) Ackroyd, H., and Hopkins, F. G. : Biochem. Jour., 1916, x, 551. 

(25) Myers, V. C, and Fine, M. S. : Jour. Biol. Chem., 1915, xxi, 389. 

(26) Osborne, T. B., and Mendel, L. B. : Jour. Biol. Chem., 1915, xx, 373. 

(27) Gelling, E. M. K: Jour. Biol. Chem., 1917, xxxi, 173. 

(28) Mathews, A. P. : Physiological Chemistry. First Edition, p. 813. 

(29) Osborne, T. B., and Mendel, L. B. : Jour. Biol. Chem., 1914, xvii, 325. 

(30) Osborne, T. B., and Mendel, L. B. : Jour. Biol. Chem., 1915, xx, 351. 

(31) Osborne, T. B., and Mendel, L. B. : Jour. Biol. Chem., 1916, xxv, 1. 

(32) Osborne, T. B., and Mendel, L. B. : Jour. Biol. Chem., 1916, xxvi, 293. 

(33) Hamilton, T. S., Grindley, H. S., and Nevens, W. B. : Unpublished 

manuscript. 

(34) Withers, W. A., and Fraps, G. S.: Bui. 179, 1901, N. Car. Agr. Exp. Sta. 

(35) Hart, E. B., and Bentley, W. H. : Jour. Biol. Chem., 1915, xxii, 477. 

(36) Grindley, H. S., and Eckstein, H. C. : Jour. Biol. Chem., 1919, xxxvii, 

373; Science, 1915, xlii, 70. 



Reprinted from Jouhnal or Dairy Science 
Vol. IV, No. 6, November, 1921 



THE PROTEINS OF COTTONSEED MEAL 1 
II. NUTRITIVE VALUE 

W. B. NEVENS 
Department of Animal Husbandry, University of Illinois, Urbana, Illinois 

I. REVIEW OF THE PREVIOUS WORK ON THE NUTRITIVE VALUE 
OF THE PROTEINS OF COTTONSEED MEAL 

Cottonseed meal and flour were found by Richardson and 
Green (1) to be satisfactory sources of protein for the growth 
of albino rats when these feeds furnished 18 per cent or more 
protein to the ration. Mendel (2) states that normal growth 
has been secured for considerable periods when the globulin of 
cottonseed was fed in suitable concentration, such concentra- 
tion having been determined by Osborne and Mendel (3) as 
18 per cent of the ration. The latter investigators (4) found 
that " Cottonseed flour forms a suitable adjuvant for the pro- 
teins of corn gluten," producing " satisfactory increments of 
growth" in chickens. In further studies of the value of certain 
proteins as supplements to corn gluten, these authors (5) demon- 
strated that the proteins extracted from cottonseed flour by 
sodium hydroxide solution were efficient supplements to the 
proteins of corn gluten for the growth of rats. The use of either 
the cottonseed globulin or the proteins precipitated from alkali 
extracts of cottonseed flour in an amount equal to 9 per cent of 
the ration resulted in "satisfactory growth" and when used to 
the extent of 6 per cent of the ration " considerable growth" 
was secured. This is interpreted as attesting the excellent 
quality of cottonseed proteins. McCollum and Simmonds (6) 
report the maintenance of body weights by rats fed a ration 
containing 6 per cent of protein derived from cottonseed. 

1 The results presented in this paper formed part of a thesis submitted to the 
Graduate School of the University of Illinois in partial fulfillment of the require- 
ments for the degree of Doctor of Philosophy in Animal Husbandry. 

552 



THE PROTEINS OF COTTONSEED MEAL 553 

In studies of the relation of the quality of proteins to milk 
production, Hart and Humphrey (7) found an equality in effi- 
ciency of the proteins of gluten feed, oil meal, distillers' grains 
and cottonseed meal as supplements to the proteins of corn 
meal and alfalfa hay. In later experiments (8) of the same 
nature, cottonseed meal proteins proved less efficient than the 
proteins of gluten feed, oil meal and distillers' grains. In these 
experiments, however, the proteins of the feedingstuff tested 
formed but 40 per cent or less of the protein content of the ration, 
and the results were calculated upon the basis of the total nitro- 
gen absorbed by the animals. 

The digestibility of the proteins of cottonseed meal is stated 
by Fraps (9) to be 88.4 per cent in the case of steers and sheep; 
by Henry and Morrison (10) as 84 per cent, for choice and prime 
cottonseed meal; by Mendel and Fine (11), who employed dogs 
as experimental animals, as 67 to 75 per cent compared to 88 to 
93 per cent for the proteins of meat; by Rather (12), using men 
as subjects, as 77.6 per cent in contrast to 96.6 per cent for the 
proteins of meat; and by Pomaski (13), who employed the gastric 
juice of the dog, as 99 to 100 per cent. 

From a review of the literature, it is apparent that investi- 
gations upon the nutritive value of the proteins of cottonseed 
meal are quite limited in extent. In the majority of experiments 
cited, the investigators drew their conclusions from the main- 
tenance of live weight, increase in live weight, state of health or 
combinations of these criteria. In most cases the amount of 
feed consumed is not recorded, so that it is impossible to judge 
whether or not the results secured were due to a failure of the 
animals to consume a sufficient amount of feed to cover their 
energy requirements. In but one series of experiments (7, 8), - 
were the conclusions based upon metabolism studies. Hence, 
the further study of the nutritive value of the proteins of cot- 
tonseed meal constituted the object of the present investigation. 

The toxicity of cottonseed meal 

Before proceeding with the investigation, it was considered 
advisable to determine, so far as possible, whether or not the 



564 W. B. NEVENS 

toxic principle of cottonseed meal is associated with its proteins, 
and further, whether cottonseed meal would prove injurious 
to rats as has been found (14) in the case of many other species 
of animals. 

From an examination of the literature, it would seem that 
there is but little basis for attributing the toxicity of cottonseed 
meal to its proteins. The assumption that the high protein 
content of cottonseed meal is responsible for its harmful effects 
(15) was denied by Dinwiddie (16), who maintains that this 
theory is not supported by a study of the recorded feeding tests. 
Withers and Brewster (17) attributed the toxic principle of cot- 
tonseed meal to a certain group of the protein molecule which 
contains loosely bound sulphur, but later work by Withers and 
associates (18) led them to conclude that the toxicity is due to 
the presence of " gossypol, " a definite chemical compound soluble 
in ether and aniline. They believe ' ' gossj^pol" may be changed to 
a nearly related substance " D-gossypol, " the latter being in- 
soluble in ether but soluble in aniline. When in alcoholic solu- 
tion either of these compounds forms precipitates with the alco- 
hol soluble proteins of wheat flour and of cottonseed meal. They 
reason that the reduction of the toxicity of cottonseed meal by 
heating may be due to the inability of the animal to digest the 
"gossypol" and " D-gossypol" protein compounds. The theory 
that gossypol is responsible for " cottonseed meal injury" is 
strengthened by the work of Alsberg and Schwartz (19). 

In their series of feeding experiments with albino rats, Rich- 
ardson and Green (1) and Osborne and Mendel (5) observed no 
toxic effects, but the cottonseed kernels themselves proved 
toxic. 

In the light of the foregoing discussion, it seems very doubtful 
if the toxicity of cotton seed meal may be attributed to either 
its high protein content or to the character of the proteins which 
it contains. Further, it seems clear that commercial cottonseed 
meal of good quality may provide practically the entire nitrog- 
enous components of the ration for albino rats over a con- 
siderable period of time with no injurious effects becoming 
manifest. 



THE PROTEINS OF COTTONSEED MEAL 555 

II. METHODS EMPLOYED IN STUDYING THE NUTRITIVE VALUE 
OF THE PROTEINS OF COTTONSEED MEAL 

Object of feeding experiment. The object of this phase of the 
experiment was to study the nutritive value of the proteins of 
cottonseed meal and to compare their nutritive value with that 
of the proteins of corn and alfalfa hay for the growth of young 
albino rats. It was planned to feed rations containing a medium 
amount of protein, derived from the above mentioned sources, 
and by means of metabolism studies to determine the extent 
to which the proteins are utilized for maintenance and growth. 

General plan of experiment. Young male albino rats in vig- 
orous, healthy condition and having an initial weight of from 
100 to 140 grams were employed. The metabolism periods were 
each seven days in length, two such periods following each other 
without intermission with each of the experimental rations 
tested. Before the first metabolism period and whenever the 
rations were changed, a three day preliminary or transition period, 
during which the ration to be employed during the metabolism 
period was fed, was inserted. It was planned to feed the ani- 
mals as large amounts of the rations as they would consume, 
the daily feed allotment being slightly greater than the amount 
consumed. 

The rats were placed in individual glass crystallizing dishes 
1\ inches in diameter and 3f inches in depth, inside measure- 
ments. The dishes were provided with weighted wire covers 
to which were attached large test tubes fitted with rubber stoppers 
and bent glass tubing, the latter extending downward through 
the wire cover. The test tubes were kept supplied with ammonia- 
free water. Large porcelain crucibles for receiving the feed 
were supported from the covers by means of wire frames. Crys- 
tallizing dishes of 60 mm. diameter were employed instead of 
the crucibles for rations containing alfalfa, which were very bulky. 
Ventilation was provided by means of a system of rubber tubes 
which conducted a current of compressed air to the bottom of 
each dish. From two to three sheets of filter paper, cut to fit 
the dishes, were placed in the bottom of each dish daily to 
absorb the urine. 

THE JOURNAL OF DAIRY SCIENCE, VOL. IV, NO. 6 



556 W. B. NEVENS 

Feces and urine were collected daily. In most cases the filter 
paper absorbed the urine completely, so that the feces were nearly 
always found dry. In a very few cases, particularly with ra- 
tions containing alfalfa which resulted in the production of 
very bulky feces, there was evidently absorption of urine by the 
feces, so that it was necessary to extract the feces once or twice 
with hot acidified water before collecting them. The feces were 
preserved under 95 per cent alcohol acidified slightly with sul- 
furic acid. At the end of each seven-day metabolism period, the 
feces were transferred to large Kjeldahl flasks and digested 
according to the to the Kjeldahl-Gunning-Arnold method with 
sulfuric acid, sodium sulfate and mercury. The resulting solu- 
tions were transferred to 500 cc. volumetric flasks and aliquots 
taken for distillation. 

After collecting the feces, the urine was extracted from the 
filter papers by washing with a stream of ammonia-free water 
acidified with sulfuric acid and held at nearly boiling tempera- 
ture. The filter paper was thoroughly pulped and pressed 
out after each extraction by means of a glass rod. From four 
to six extractions were made, using 40 to 60 cc. of water each 
time, the sides and bottom of the dish also being thoroughly 
washed. The extracts were filtered through glass wool into 
250 cc. volumetric flasks. The flasks were allowed to remain in 
the ice box over night. The solutions were then made up to 
volume at ice box temperature and transferred to 2.5 liter bottles 
which were kept in a cold storage room at a temperature of 
5° to 10° C. until analyzed. About 0.5 gram of powdered thymol 
was employed as a preservative in each bottle in which the week's 
urine was collected. The composites were thoroughly mixed and 
aliquots measured out in the cold for total nitrogen determi- 
nations. 

The feed was weighed daily into the crucibles and mixed with a 
little nitrogen-free water to the consistency of a thick paste. 
The following day the feed residues were scraped out and dried 
in the same oven and at the same temperature as the rations 
used. In some cases the animals scattered the feed from the 
crucibles about the metabolism dish. In such cases the eed 



THE PROTEINS OF COTTONSEED MEAL 557 

remaining in the metabolism dish at the time of collecting the 
excreta was carefully separated and added to the feed residues. 
When thoroughly dry the weight of the feed residues was de- 
termined and the amount of feed actually consumed during the 
seven-day period calculated. By previous tests in this labora- 
tory it was found that the error involved in this calculation due to 
a difference in the moisture content of the residues and ration 
was less than 1 per cent, and further that the nitrogen contents 
of the residues and ration were identical (20). 
■ Preparation of rations. In preparing the experimental rations, 
the starch used was first dextrinized by heating on the steam 
bath after the addition of cold water and a few crystals of citric 
acid. When ground corn formed one of the constituents of a 
ration, it was mixed with the starch and the starch of the mix- 
ture dextrinized. The other ingredients were then added, the 
agar being dissolved in boiling water and added at the boiling 
temperature. When necessary more hot water was added and 
the ingredients thoroughly mixed. The rations were dried on 
glass plates, placed above the steam bath, finely ground and 
dried in an oven at a temperature of about 40°C. After drying 
for several days, the rations were mixed, sampled for analysis 
and placed in tightly covered glass jars. 

The nitrogen free ration consisted of the following: 

per cent 

Salts. 5 

Butterfat 10 

Sucrose 8 

Starch. 74 

Agar 3 

Water soluble vitamin, 150 mgm. of solids per 100 grams of ration. 

The composition of the other rations is shown in table 1. The 
salt mixture used was compounded according to the formula of 
Osborne and Mendel (21), while the water soluble vitamin 
consisted of Osborne and Wakeman's (22) fraction II of the 
concentrated extract of the water soluble vitamin of brewers' 
yeast. The stock supply of the latter was prepared in the form 
of a water solution which was preserved by means of a small 
quantity of chloroform and kept in the ice box. The butter- 



558 



W. B. NEVENS 



fat was obtained by placing fresh creamery butter in large 
beakers, heating to a temperature of 50° to 60° on the steam 
bath, centrifuging for an hour or until the fat became water 
clear, and then siphoning off the clear fat. 

It was planned that all rations containing a protein feeding- 
stuff should carry 10 per cent of protein (N X 6.25), but the 
actual content of protein was slightly higher, ranging from 10.38 
per cent to 11.28 per cent, due to the fact that some of the con- 
stituents used in making up the ration had a slightly higher 
moisture content than the dried rations. 

TABLE 1 
Composition of experimental rations (expressed in percentage) 



CONSTITUENT 


EATION 




1* 


2 


3 


4* 


5 


6 


7 


Cottonseed meal 


23.9 

55.1 
3.0 
5.0 

10.0 
3.0 


72.7 

6.3 
3.0 
5.0 
10.0 
3.0 


63.5 
18.5 

5.0 

10.0 

3.0 


13.1 
32.3 

33.7 
3.0 
5.0 

10.0 
3.0 


28.4 
40.6 
13.0 

5.0 

10.0 

3.0 


10.3 

35.7 
36.0 

5.0 

10.0 

3.0 


7.7 


Corn 


19.3 


Alfalfa hay 


29.3 


Starch 


25.7 


Agar 




Sucrose 


5.0 


Butterfat 


10.0 


Salts 


3.0 






Total 


100.0 
1.750 


100.0 
1.660 


100.0 

1.806 


100.0 

1.777 


100.0 
1.708 


100.0 
1.790 


100.0 


Total nitrogen content. 


1.782 



* Water soluble vitamin preparation added at the rate of 1.5 mgm. of solids 
per gram of ration to rations 1 and 4. 



In preparing the rations in which two or more feedingstuffs 
were combined an effort was made to have each feedingstuff 
furnish an equal amount of digestible protein, using the coeffi- 
cients of digestibility secured in period 2 as a basis for calcula- 
tion, but keeping the total content of crude protein the same 
throughout the experiment, namely, 10 per cent. 

The cottonseed meal used in the rations was a part of the same 
sample which was employed in the analytical study presented 
in a preceding paper. Through the courtesy of the Plant Breed- 
ing Division of the Agronomy Department of this university a 



THE PROTEINS OF COTTONSEED MEAL 559 

quantity of "high protein" corn containing 2.2 per cent nitro- 
gen was secured, which made it possible to formulate a suitable 
corn ration containing 10 per cent protein. All of the feeding- 
stuffs used were in a finely ground condition before compounding 
the rations. 

Accuracy of metabolism work with small animals. Since the 
accuracy of metabolism work depends in large measure upon the 
accuracy of the collection of the excreta, especially when such 
small amounts of nitrogen are involved as in the case of the 
smaller laboratory animals, several experiments were carried 
out to test the accuracy of the methods employed. 

Each day during period 6, when 6 rats were receiving the same 
feed mixture, the paper residues remaining after extraction of 
the urine were collected in glass jars and placed in the ice box. 
At the end of the metabolism period, the entire mass of residues, 
including the glass wool used in filtration, was transferred to a 
2 liter beaker and boiled for some time with about 1 liter of 
water acidified with sulphuric acid. The extracts were decanted 
and the procedure repeated, the acidified water being pressed 
out from the residues. The extracts were filtered through glass 
wool, evaporated on the steam bath and transferred to Kjeldahl 
flasks for total nitrogen determination. The paper residues 
also, together with the glass wool, were transferred to large 
Kjeldahl flasks, and total nitrogen determined. The results of 
these determinations are shown in table 2. The nitrogen ex- 
tracted in the procedure described just above is assumed to be of 
urinary origin and is compared to the total amount of urinary 
nitrogen excreted during the week. It is shown that, as an 
average of 6 such determinations, the error in the collection of 
urine amounted to 2.0 per cent of the total nitrogen. 

The nitrogen remaining in the paper residues which was not 
extracted by boiling with acidified water was assumed to be 
fecal nitrogen. In collecting the feces it was sometimes impos- 
sible to entirely remove the fecal matter from the filter papers, 
especially when the rations tended to cause a laxative condition. 
Such a condition was not a constant effect with any ration em- 
ployed, but was more frequent with rations containing alfalfa. 



560 



W. B. NEVENS 



With the latter rations, three filter papers were generally placed 
in each dish daily, while with the other rations two papers were 
used. Analysis of the filter paper showed that seven filter papers 
of the size used contained 1.06 mgm. of nitrogen. In making 
the calculations shown in table 2 it was assumed that the nitro- 
gen originally contained in the filter papers was insoluble in the 
hot dilute acid employed in extraction and this has been de- 
ducted from the non-extractable nitrogen remaining in the 
paper residues. To the extent that such nitrogen is soluble in 



TABLE 2 
Test of the completeness of extraction of nitrogen from filter papers used as 
absorbents during one metabolism period 





fc 


U 


£ 


u 


63 Pj 


>j 


£ 


o 


K 


K 63 


a 




63 £ 


t> 




3 fc; 


01 P 
< P- 


o 


►3 


►3 fk 
►J 63 




63 Ph 


pj 




m 




< 
5« 


85 






63° 


O o 

O 63 


111 s 

*a 


O O 


K o 


RAT NUMBER 


H 63 
O Hi 


P o 






P2 




Eh 

63 




2 Pi 


Z 






<! < 

m p< 


RR 




PS 5, 

2 o 


ff & 


fc B 




P5 17 

2o 


« p. 


*d eh 1 a 


3 3 




K § 


" 63 


5 « 


K Eh 


s* 


" 63 


O 63 


P3 S 


En P 
O Ph 


Eh PS « 
O 3 K 


&8 




63 


H 


63 


z 


Eh 


63 


Eh ■ 


Eh 


Eh 




mgm. 


mgm. 


mgm. 


per 
cewi 


mgm. 


mgm. 


mgm. 


per 
cent 


mpm. 


mgm. 


per 

cent 


2 


12.5 


558.4 


570.9 


2.2 


25.5 


546.8 


572.3 


4.5 


1143.2 


38.0 


3.3 


3 


15.9 


716.3 


732.2 


2.2 


25.8 


833.7 


859.5 


3.0 


1591.7 


41.7 


2.6 


5 


9.0 


619.3 


628.3 


1.4 


22.2 


645.3 


667.5 


3.3 


1295.8 


29.2 


2.3 


6 


13.2 


541.0 


554.2 


2.4 


20.1 


615.8 


635.9 


3.2 


1190.1 


33.3 


2.8 


8 


11.5 


652.5 


664.0 


1.7 


12.9 


672.0 


684.9 


1.9 


1348.9 


24.4 


1.8 


9 


18.3 


764.3 


782.6 


2.3 


28.0 


602.0 


630.0 


4.4 


1412.6 


46.3 


3.3 


Average 


13.4 




655.4 


2.0 


22.4 




675.0 


3.3 


1330.4 


35.5 


2.7 







* After deduction of nitrogen contained in same number of clean filter papers. 

hot dilute acid, however, it would tend to offset to a small degree 
the losses in the collection of urine, although the correction would 
not be in proportion to the variable total urinary nitrogen. The 
error in the collection of feces was found to be 3.3 per cent, using 
the average of six determinations, or, comparing the total nitro- 
gen lost to the total nitrogen excreted in urine and feces during 
the week, the total error in the collection of both feces and urine 
is 2.7 per cent. 

The results obtained in this test were applied to the metabolism 
data for period 6, the period during which this test was conducted, 



THE PROTEINS OF COTTONSEED MEAL 561 

to determine what effect the incomplete collection of the ex- 
creta has upon the utilization coefficients. It is evident that 
an error in the collection of the excreta is reflected directly in 
the nitrogen balance and in the percentage utilization. Using 
the average values given in table 2 of 13.4 mgm. of nitrogen 
representing uncollected urine and 22.4 mgm. of nitrogen repre- 
senting uncollected urine and 22.4 mgm. of nitrogen representing 
uncollected feces during a seven day period, and applying them 
to the data for the individual animals during period 6 it is found 
that the percentages of utilization of absorbed nitrogen as given 
in the tables are approximately 2 per cent too high, while the 
percentages of absorbed nitrogen retained are slightly more than 
3 per cent too high. Like results are obtained for period 7 
during which the animals received the same ration as in period 6. 

The completeness of the collection of urine was tested in 
still another way, that of the recovery of urea which was added 
in the form of a standard solution to the daily feed. Two mature 
rats were employed in the test. After the excretion of urinary 
nitrogen had been reduced to a nearly constant level by sub- 
sistence on protein free rations- for seven days, known amounts 
of urea were added to the ration. 

The excretion of this extra nitrogen was very prompt, as indi- 
cated by the results of the test as shown in table 3. In the 
case of rat 10, the results are somewhat difficult to interpret, 
owing to the fact that the consumption of feed decreased rapidly, 
evidently resulting in catabolism of small amounts of body pro- 
tein to furnish energy for the body. By using the figures for 
the average excretion of nitrogen during the three days prelimi- 
nary to the first urea day as the level of the endogenous nitro- 
gen during the three urea days, the apparent recovery of the 
urea nitrogen amounted to 119 per cent. Similar results are 
obtained if the average nitrogen excretion during the prelimi- 
nary and subsequent periods are employed. If it be assumed that, 
owing to a decrease in feed consumption below that of the energy 
requirements, the endogenous nitrogen should be taken as cor- 
responding to that in the subsequent period, then the recovery 
of the urea nitrogen was approximately 100 per cent. Undue 



562 



W. B. NEVENS 



emphasis should not be placed upon the results given by this 
animal, however. 

With rat 11 more reliable data were obtained as it was not 
evident that the feed consumption Was deficient in meeting 
the energy requirements. By using the average nitrogen excre- 
tion during both preliminary and subsequent periods as the 
level of the amount of body nitrogen excreted, the recovery of 

TABLE 3 
Test of the completeness of collection of urine by addition of urea to the ration 



LIVE WEIGHT FEED EATEN UREA N ADDED DAILY URINARY N 



RatlO 





grams 


grams 


mgm. 


mgm. 


1 


182 


10.5 





35.6 


2 




10.5 





24.0 


3 




10.5 





23.5 


4 


178 


6.1 


42.1 


74.6 


5 




6.1 


42.1 


83.0 


6 




6.1 


42.1 


76.0 


7 




4.6 





35.9 


8 


167 


5.6 





35.8 



Rat 11 



1 


176 


7.2 





42.8 


2 




7.2 





30.2 


3 




7.2 





39.8 


4 




7.7 


56.5 


82.8 


5 




7.7 


56.5 


101.8 


6 




5.4 





37.5 


7 


164 


5.4 





29.3 



urea nitrogen amounted to 95 per cent of that fed. If the aver- 
age of the amounts of nitrogen excreted during days 3 and 6 be 
used as this level, then the recovery of urea nitrogen was prac- 
tically 100 per cent. 

It is evident from the data presented concerning the recovery 
of urea nitrogen that the method for the collection of urine as 
employed in these experiments, gives very nearly quantitative 
results. 



THE PROTEINS OF COTTONSEED MEAL 563 

Further tests of the metabolism method employed, which were 
performed in this laboratory and are described below, show that 
loss of ammonia due to bacterial decomposition of the urine does 
not occur to any appreciable extent. 

In the first test, three portions of urine of 5 cc. each were 
measured out for total nitrogen determination. At the same 
time 5 cc. portions of urine were added to each of six metabolism 
dishes containing the usual number of filter papers. Three of 
these dishes were allowed to stand at room temperature in the 
metabolism laboratory, while the remaining three were placed 
in an oven at a temperature of about 40°C. At the end of twenty- 
four hours, the urine was collected from all six dishes in the same 
manner as employed in the metabolism work, i.e., by washing 
with hot acidified water. As a result of this test it was found 
that 5 cc. of urine contained 28.18 mgm. of nitrogen, while the 
amounts of nitrogen recovered from the dishes kept for twenty- 
four hours at room temperature and at 40°C. were, respectively, 
27.61 mgm. and 27.54 mgm. 

In the second of these tests, the urine from one rat receiving a 
constant amount of the same ration was collected daily. On the 
first, third, and fifth days the urine was collected at once by 
washing with acidified water in the usual manner. The urine was 
made up to a volume of 250 cc. and aliquots taken at once for 
total nitrogen determinations. 

On the alternate days the urine was not collected at the end of 
the twenty-four hour period, but the filter paper was moistened 
and the dish allowed to stand another twenty-four hours in the 
metabolism laboratory before extraction in the usual maner. 
The rat meanwhile was transferred to a clean dish. At the end 
of the second day the urine was collected by washing as usual, 
made up to volume, and aliquots taken for total nitrogen deter- 
mination. The results of the test are shown in table 4. It is 
evident that there was no appreciable loss of nitrogen due to 
bacterial decomposition even after the metabolism dishes had 
stood for two days. 

How shall the nutritive value of proteins be compared? In 
attempting to compare the biological values of various feeding- 



564 



W. B. NEVENS 



stuffs, it is first necessary to select a suitable basis for compari- 
son. Several different methods for comparison are in use. As 
pointed out in the introduction one of the most common methods 
is to base conclusions upon the character of the growth secured, 
the principal index in such a case being the gain in live weight. 
Some of the data from table 1 of the appendix are brought 
together in table 5. These data were all obtained in periods 2 
and 3. The rats consuming the cottonseed meal ration showed 
marked fluctuations in gain in live weight which can not be ac- 
counted for on the basis of a variable food intake. With the 
corn ration, there was a gain in weight by one rat in one period 



TABLE 4 



Effect of allowing metabolism dishes to stand twenty-four hours and forty-eight 
hours before the collection of urine 







DAILY URINARY N WHEN 


DAILY URINARY N WHEN- 


DAY OP 


EXPERIMENT 


COLLECTED AT END OP TWENTY- 


COLLECTED AT END OF FORTY- 






FOUR HOURS 


EIGHT HOURS 






mgm. 


mgm. 




1 


57.9 






2 




64.9 




3 • 


60.8 






4 




67.3 




5 


63.2 






6 




60.3 


Average 




60.3 


62 1 







only. The nitrogen of the ration, however, was being used by 
the body to a considerable extent, for on a nitrogen-free ration 
the same animals lost 16 to 19 grams in weight during a period 
of equal length, compared to 1 to 2 grams on the corn ration. 
Likewise, there was also a large variation in gains in weight by 
the rats receiving the alfalfa ration, the average gains of rats 
7 and 8 being almost zero. It is probable that many factors 
other than the quality and amount of the protein consumed 
influence the gain in weight, such as the proportion of carbohy- 
drates in the ration, amount of water drank, exercise, the pro- 
portions of gain which is protein or fat, etc. While interpreta- 
tions based upon the gain in live weight may lead to reliable 



THE PROTEINS OF COTTONSEED MEAL 565 

conclusions in some instances, the adoption of such a criterion 
in the present case would certainly be a fallacious procedure. 

It is a matter of common knowledge that all animals require 
protein food for keeping the body tissues intact, known as the 
maintenance requirement, and secondly, that growing animals 
need an additional quantity of protein for the construction 
of new tissue. If a standard for the comparison of the value of 
the proteins of feedingstuff s for growth is based simply upon the 
proportion of the nitrogen of the feedingstuff which is retained 
by the body, the values secured in such a manner are subject 
to gross errors. With such a method of computation the ap- 
parent value of the proteins for growth depends largely upon the 
nitrogen intake, or, in other words, upon the amount of feed 
eaten, and this in turn is subject to individual idiosyncracy and 
the palatability of the ration. As mentioned elsewhere, when 
the ration proves unsatisfactory, rats tend to eat less and less 
from day to day. By reference to table 5 it may be seen that 
both rats 5 and 6 ate less of the corn ration during period 3 
than during period 2. Rat 5, during period 2, retained 16 per 
cent of the nitrogen absorbed, but during period 3, when the 
amount of feed consumed was evidently too little to maintain 
the animal's live weight, the nitrogen of the excreta was greater 
than the nitrogen intake, so that there was a loss of nitrogen from 
the body resulting in a negative value for the percentage of 
absorbed nitrogen retained. Similarly, the percentage of ab- 
sorbed nitrogen retained by rat 6 falls from 23 per cent in period 
2 to 9 per cent in period 3, a change which in this instance may 
also be attributed to a decreased food intake. Were the average 
percentage of the absorbed nitrogen retained by rats 5 and 6 
taken as a measure of the utilization of the proteins of corn for 
growth, it would be a distorted picture of the facts. 

There seem to be factors other than the amount of feed con- 
sumed which render the use of the percentage of absorbed nitro- 
gen retained an unsatisfactory criterion of the utilization value of 
the proteins of feedingstuffs. As may be seen by reference to 
table 5, the percentage of nitrogen retained by rat 7 in periods 
2 and 3 falls from 23 per cent to 8 per cent, and in the case of 



566 



W. B. NEVENS 



rat 8 the percentage falls from 16 per cent in period 2 to 6 per 
cent in period 3. These violent fluctuations are not due entirely 
to a decreased nitrogen intake, for in the case of rat 7 the 
feed intake increased slightly during the second period. They 
are, however, associated with a slightly decreased digestibility, 
although there may be other causative factors. 



TABLE 5 
A comparison of three methods of expressing the utilization of proteins 













UTILIZA- 




RAT 

NUMBER 


PERIOD 


RATION 


FEED 

CONSUMED 

DAILY 


GAIN 

IN WEIGHT 

FOR 

PERIOD 


TION OF 
ABSORBED 
N FOR 
MAINTE- 
NANCE AND 
GROWTH* 


ABSORBED 

N 

RETAINED* 








gm. 


gm. 


per cent 


per cent 


1 


2 


Cottonseed meal 


9.52 


11 


63 


31 


1 


3 


Cottonseed meal 


9.48 


6 


65 


29 


2 


2 


Cottonseed meal 


8.66 


9 


64 


21 


2 


3 


Cottonseed meal 


8.57 


5 


64 


17 


3 


2 


Cottonseed meal 


11.51 


12 


71 


44 


3 


3 


Cottonseed meal 


12.47 


11 


70 


43 


4 


2 


Corn 


7.44 


2 


49 


15 


4 * 


3 


Corn 


8.05 





47 


15 


5 


2 


Corn 


7.66 





54 


16 


5 


3 


Corn 


6.30 


-1 


43 




6 


2 


Corn 


7.49 


-1 


55 


23 


6 


3 


Corn 


6.35 


-2 


48 


9 


7 


2 


Alfalfa hay 


9.33 


6 


62 


23 


7 


3 


Alfalfa hay 


9.50 


-5 


57 


8 


8 


2 


Alfalfa hay 


9.19 


2 


58 


16 


8 


3 


Alfalfa hay 


8.20 


-2 


57 


6 


9 


2 


Alfalfa hay 


11.42 


3 


67 


22 


9 . 


3 


Alfalfa hay 


13.67 


7 


73 


38 



* For method of calculation of these percentages, see table 1 of the appendix. 

Any suitable criterion used in feeding experiments for the 
comparison of the utilization of proteins for growing animals 
must necessarily consider the effect of the proteins in providing 
nitrogen for maintenance, for in growing animals these processes 
proceed concurrently. It is doubtful if the true protein require- 
ment for the maintenance of a growing animal can be determined 
by feeding a ration containing protein, for Waters (23) has shown 



THE PROTEINS OF COTTONSEED MEAL 567 

that when young steers received a ration which just maintained 
their live weight some of the growth processes continued. Simi- 
lar results were obtained by Aron (24). 

Perhaps the nearest approach to the determination of the 
exact amount of nitrogen required for the maintenance of a 
growing animal is a study of the nitrogen excretion when the 
ration consists entirely of carbohydrates and this is being taken 
in an amount in excess of the body's energy requirement. Under 
such conditions the nitrogen excretion falls to a very low level, 
often to one third or less of that during starvation, as shown by 
Folin (25), Landergren (26), Cathcart (27) and Thomas (28). 
The amount of protein then being catabolized has been defined by 
Rubner (29) as the " wear and tear" quota of protein metabolism, 
which requires a "repair quota" of protein in the diet in order 
to replace it. A " growth quota" must be supplied the young 
animal in addition to the " repair quota" in order that growth 
may take place. Using dogs as experimental animals, Michaud 
(30) found, when protein in the form of casein or dog tissue 
was fed in amounts equivalent to the protein minimum after 
the metabolism had been reduced to this level, that there was 
no further loss of nitrogen from the body. Thomas (28) found, 
after the reduction of the nitrogen excretion by a carbohy- 
drate diet to the minimum level, that nitrogen equilibrium could 
be restored by the ingestion of an amount of protein nitrogen 
in the diet equal to the amount of nitrogen being eliminated in 
the excreta. 

On a nitrogen-free diet the amount of nitrogen excreted daily in 
the feces was about 1 gram, and this amount was not increased 
with a nitrogen intake of 3 grams furnished by a highly diges- 
tible protein. With diets producing a large bulk of feces he 
found that a greater proportion of digestive juices was elimi- 
nated, increasing the nitrogen content of the feces. 

In the interpretation of the feeding experiments which follow 
it is assumed that the amount of protein required for body main- 
tenance is a constant value for each individual at a given weight. 
Such an assumption is entirely in harmony with the theories of 
many investigators in the fields of both human and animal nu- 



568 W. B. NEVENS 

trition. Folin (25), as a result of his study of the different forms 
in which nitrogen is excreted on high and low protein diets, was 
led to formulate his theory of two distinct types of metabolism. 
The endogenous is most characteristically represented by the 
excretion of creatinine, which, "ona meat-free diet is a constant 
quantity, different for different individuals, but wholly inde- 
pendent of quantitative changes in the total amount of nitrogen 
eliminated." Folin's results have been substantiated by an 
immense amount of investigation concerning urinary creatinine, 
and his theory of protein metabolism is now almost universally 
accepted in its main essentials, although it has been necessary 
to modify this view slightly with our increased knowledge of the 
chemistry of the proteins. 

The constancy of the protein minimum for the individual is 
accepted by Lusk, Thomas and others. That this minimum 
differs between individuals and is subject to slight variation due 
to environmental, temperamental and dietary changes, possi- 
bilities which are not precluded by Folin's theory, is brought out 
by Cathcart (31) : 

As regards the uniformity of the protein minimum it may be defi- 
nitely stated that there is no single minimum — common to all men and 
to all conditions. Rubner, Caspari and others also hold firmly to this 
opinion. Caspari quotes the work of Larguier des Bancels in 1903 
in confirmation of this belief in the existence of mutiple protein minima. 
The facts that can be cited against a common minimum are many in 
number. Thus the caloric value of the diet given influences very 
materially the amount of nitrogenous material required, as is shown, 
for example, in the experiments of Voit and Korkunoff. Then, as 
Rubner has pointed out, the temperature influences quite markedly 
the course of protein metabolism. Finally, another factor of consid- 
erable importance may be mentioned, the activity of the organism. 

In the sphere of animal nutrition, the constancy of the main- 
tenance requirement for farm animals is recognized by Kellner, 
Armsby and Haecker. C. Voit and Kellner also proved conclu- 
sively that work production of varying intensity by farm animals 
does not increase the protein metabolism appreciably. 



THE PROTEINS OF COTTONSEED MEAL 569 

In this connection it should be stated that some of the cur- 
rent theories of protein metabolism are not in complete harmony 
with that just mentioned. Among these are the reversible reac- 
tion theory of Sherman (32), which seeks to account for the func- 
tions which the food protein serves in body maintenance by the 
assumption that the absorption of the amino acids liberated in 
digestion causes an increased concentration of these in the tis- 
sues which checks or even reverses the hydrolysis of tissue protein. 
This theory is hardly compatible with the known facts regarding 
the constancy of the endogenous metabolism, which has been 
found (33) to be uniform from hour to hour, as evidenced by the 
creatinine elimination, even during digestion and absorption of 
proteins. Absorption of the protein digestion products from the 
alimentary tract presumably occupies only a portion of the 
twenty-four hour period, so that even if the endogenous catab- 
olism were inhibited by the increased concentration of amino 
acids, it would be only temporary, for it has been shown (34) 
that, in adult rats, protein feeding has only a very slight effect 
upon the amino acid concentration in the tissues. This known 
slight increase in the amino acid content of the tissues during 
digestion would not be of sufficient magnitude to inhibit the 
action of digestive enzymes when a digestion experiment is 
conducted in vitro. Further, it is unreasonable to assume that 
anabolism and catabolism of tissue proteins are simply rever- 
sible phases of the same reaction and that both these processes 
are promoted by the same enzyme. In the young growing 
animal protein feeding has been demonstrated (34) to increase 
considerably the amino acid content in the tissues. Were the 
endogenous metabolism inhibited entirely during the time this 
concentration is maintained, as must be assumed from the re- 
versible reaction theory, then the catabolism of tissue protein 
per unit of weight in the young growing animal would be but a 
fraction of that of a mature animal. 

Osborne and Mendel (35) explain the maintenance protein 
requirement upon the need of certain amino acids to serve special 
physiological functions, such as the formation of the active prin- 
ciples of the internal secretions and hormones. This theory 



570 W. B. NEVENS 

assumes, therefore, that when the animal is receiving a nitrogen- 
free ration, body tissue must be catabolized to furnish the essen- 
tial amino acids, but that, on the other hand, when a ration con- 
taining a complete assortment of amino acids in sufficient amount 
is being consumed the endogenous metabolism is only a frac- 
tion of that on a non-nitrogenous ration, and that then only 
the catabolism of the internal secretions or the tissues which 
regulate metabolism would be affected. Under these condi- 
tions the muscles would scarcely be affected and the creatinine 
elimination would bear little relation to the endogenous metab- 
olism. Moreover, the theory does not satisfactorily account 
for the effect of ammonium salts, mixtures of amino acids and 
single amino acids in partially supplying nitrogen for main- 
tenance. 

Since the plan of procedure and method of calculation employed 
in this investigation are dependent primarily upon the basic 
assumption that the endogenous metabolism of the animal or- 
ganism is constant in character and amount for an individual 
at a given age and weight, an examination of the data obtained 
during all the metabolism periods was made in order to ascer- 
tain, if possible, whether this assumption is substantiated by 
the experimental results at hand. In making this examination, 
the data embodied in appendix table 1 were employed to obtain 
the first set of values shown in the column headed "As deter- 
mined" under each "period" of table 6. These values were 
obtained by deducting the sum of the endogenous nitrogen and 
the metabolic nitrogen in the feces from the daily urinary nitro- 
gen and calculating the percentage of the daily nitrogen intake 
which the remainder forms. The second column of values under 
each "period, " headed "As calculated, " was obtained in the same 
way as those in the first column, except that the average value 
for daily urinary nitrogen as determined with nine rats in period 
1, i.e., 22 mgm. per 100 grams live weight, is used in calculating 
the "endogenous nitrogen" for periods 2 to 7, inclusive, instead 
of the individual values determined in the same period. 

It is shown in table 6 that the individual daily urinary nitrogen 
values for rats 2, 5, and 9 are above the average, while those for 



THE PROTEINS OF COTTONSEED MEAL 



571 



rats 3, 6 and 8 are below the average. Hence, for comparison, 
rats 2 and 3, 5 and 6, and 8 and 9 are arranged in pairs. The 
rats of each pair received the same rations throughout the ex- 
periment. It is natural to assume that two animals of the 
same age and weight and in a comparable nutritive condition 
will utilize the same ration with an equal degree of efficiency, 
subject of course to inherent individual variability. By refer- 
ence to table 6, it may be noted that this holds true to a very 
great extent, although the natural variations to be expected in 
biological work of this kind are in evidence. 



TABLE 6 



The proportion of the daily intake of nitrogen above maintenance which appears 
in the urine (expressed in percentage) 





PERIOD 2 


PERIOD 3 


PERIOD 4 


PERIOD 5 


PERIOD 6 


PERIOD 7 


RAT NUMBER 


As 
deter- 
mined 


As 
calcu- 
lated 


As 
deter- 
mined 


As 
calcu- 
lated 


As 
deter- 
mined 


As 
calcu- 
lated 


As 
deter- 
mined 


As 
calcu- 
lated 


As 
deter- 
mined 


As 
calcu- 
lated 


As 
deter- 
mined 


As 
calcu- 
lated 


2 


12.0 


16.3 


9.8 


13.1 


1.7 


5.3 


8.3 


12.4 


8.9 


13.4 


8.8 


13.7 


3 


8.8 


8.3 


9.4 


8.9 


7.8 


7.2 


7.3 


6.9 


9.9 


9.3 


9.9 


9.2 


5 


26.5 


29.1 


35.0 


38.2 


18.7 


21.0 


18.5 


21.1 


12.3 


14.4 


11.4 


13.7 


6 


23.9 


20.0 


31.3 


26.7 


21.0 


17.5 


16.4 


12.3 


12.6 


9.5 


14.0 


11.1 


8 


9.1 


7.4 


7.8 


5.8 


15.6 


14.3 


10.4 


8.9 


14.3 


12.5 


9.7 


7.7 


9 


4.1 


5.5 


1.9 


3.1 


15.8 


17.3 


14.9 


16.4 


16.0 


17.6 


16.4 


18.1 



Considering first the values listed under the headings "As 
determined" in each period it is evident that there is a marked 
uniformity exhibited by the animals of each pair throughout the 
different experimental periods with but few exceptions. For 
example, it is shown that rats 2 and 3 eliminate in the urine 
about the same proportion of the nitrogen intake above main- 
tenance "as determined," with the exception of periods 2 and 
4. Rats 5 and 6 show quite uniform results throughout the 
entire six periods. Rats 8 and 9 do not very widely from each 
other in periods 4, 5 and 6. Moreover, certain rations seem to 
have a pronounced effect on these percentages. Rats 5 and 6 
received the corn ration during periods 2 and 3, and the corn- 



572 W. B. NEVENS 

cottonseed meal ration during periods 4 and 5. With both of 
these rations, the proportion of the daily nitrogen intake elimi- 
nated in the urine was greater than with the other rations, but 
both animals behaved alike in this respect. 

On the other hand, when the average values for the endoge- 
nous nitrogen of the urine are used in calculating maintenance, the 
variations in the percentages of the nitrogen intake above main- 
tenance appearing in the urine of the animals of each pair are 
greatly exaggerated in 14 of the 18 cases involved, as shown under 
the headings "As calculated." In one of the four cases, that of 
rats 5 and 6, period 7, there is no change. In the other three 
cases, those of rats 2 and 3, period 4, and rats 8 and 9, periods 
2 and 3, the spread is lessened slightly. In many cases the use 
of an average maintenance factor increases the spread between 
the animals of a pair as much as 200 per cent. These data seem 
to indicate quite conclusively that the endogenous metabolism 
is a function of the individual animal and that this is a definite 
and probably constant value under a given set of conditions. 
This is quite in harmony with the theory of Folin (25) respecting 
the constancy of endogenous metabolism. In calculating the 
results of these experiments, therefore, the use of individual 
maintenance values is evidently justifiable. 

If, having determined the minimal "wear and tear" quota of 
an animal by appropriate metabolism experiments, feeding 
tests are then initiated to study the utilization of the proteins of 
feedingstuffs by that animal, it is possible to calculate the pro- 
portion of the nitrogen excreted in the urine which is of endoge- 
nous origin and likewise the amount of fecal nitrogen whose source 
is metabolic. This method follows closely that of Thomas (28) 
in calculating the biological values of foodstuffs. Such a criterion 
evaluates the proportion of the nitrogen of the food which is 
actually utilized by the animal in its metabolism, whether the 
animal is consuming an amount of protein which is not quite 
sufficient for it to maintain its live weight, or whether growth is 
permitted. The values obtained by applying this method of 
calculation to the data of metabolism periods 2 and 3 are shown 
in the last column of table 5. An inspection of these values 



THE PROTEINS OF COTTONSEED MEAL 573 

shows that this method overcomes some of the objections raised 
to the other methods. When the gain in live weight falls to 
zero or a little below, but at the same time it is evident that some 
of the nitrogen of the ration is being used by the animal, the 
percentage " utilization of the absorbed nitrogen for mainte- 
nance and growth" is lowered, but is, nevertheless, a very dis- 
tinct positive value. With a slight decrease in food intake, there 
is usually a corresponding fall in the ''utilization of absorbed 
nitrogen for maintenance and growth" coefficient, but this 
decrease is not so extreme as when the results are calculated upon 
the basis of the " absorbed nitrogen retained." Moreover, a 
decrease in the feed intake to just below the maintenance level 
does not result in a negative value. On the whole the "utiliza- 
tion of absorbed nitrogen for maintenance and growth" coeffi- 
cients are much less variable than those of the " absorbed nitro- 
gen retained." The former method is subject to a coefficient 
of variability of 8.4 per cent when all of the values obtained in 
the six metabolism periods are considered, and a mean is assumed 
for each ration. Similarly, the same values when calculated 
upon the basis of the percentage of " absorbed nitrogen retained" 
have a coefficient of variability of 27.7 per cent, a striking and 
important difference. 

In this investigation, therefore, the endogenous metabolism 
of the experimental animals was studied during a metabolism 
period in which a nitrogen-free ration was fed, and this was fol- 
lowed by six metabolism periods in which the proteins of cot- 
tonseed meal were compared with those of corn and alfalfa hay. 

III. DISCUSSION OF THE RESULTS 

Metabolism of the rat on a nitrogen-free ration. During the 
first eleven days of the experiment the rats which had been con- 
suming an ordinary stock ration were given nitrogen-free rations 
prepared as described above. On the fifth day collection of the 
feces and urine was begun, and continued for seven days. In 
order to check the results secured during the first period of the 
experiment, three of the rats were again placed on nitrogen-free 



574 



W. B. NEVENS 



rations during period 6, after having received nitrogenous rations 
during the intervening time. The principal data of these trials 
are included in table 7. The rats lost slightly more than 2 grams 
of weight per head daily. For the first few days of the period the 
animals ate the ration in large quantities, but as they apparently 
found the feed unsatisfactory, they consumed smaller and smaller 

TABLE 7 
Metabolism of the rat when receiving a nitrogen free ration 



RAT 

NUMBER 


PERIOD 


INITIAL 
WEIGHT 


FINAL 
WEIGHT 


AVERAGE 

FEED 

CONSUMED 

DAILY 


DAILY 
URINARY 
NITROGEN 


DAILY 

FECAL 
NITROGEN 


DAILY 
URINARY 

N PER 

100 GRAMS 

LIVE 

WEIGHT 


FECAL 
NITROGEN 

PER 

100 GRAMS 

FEED 


1 

2 
3 

4 
5 
6 

7 
8 
9 




grams 

99 
118 
134 
135 
120 
129 
123 
122 
126 


grams 

88 
102 
116 
118 
104 
110 
107 
109 
114 


grams 

6.51 

7.43 
9.07 
8.79 
7.29 
7.86 
7.43 
8.14 
7.43 


mgm. 

25 
31 
26 
24 
28 
21 
23 
22 
29 


mgm. 

17 
19 

24 
22 
20 
21 
20 
24 
18 


mgm. 

27 
28 
21 
19 
25 
18 
20 
19 
25 


mgm. 

253 
255 
259 
251 
273 
270 
266 
298 
246 


Average 








7.78 






22 


264 


1 

4 

7 


6 
6 
6 


113 
135 

118 


102 
130 
105 


6.10 
9.02 
4.76 


22 
16 
24 


19 
20 
17 


20 
15 
21 


306 
223 
357 


Average 








6.63 






19 


295 



amounts from day to day, and a few of the animals scattered the 
feed from the containers at once upon being fed. 

It was found that, while subject to some individual variation, 
the amount of urinary nitrogen per 100 grams of live weight is 
fairly constant, the average value of 22.4 mgm. obtained agreeing 
almost exactly with that found by Mitchell (20) in a large num- 
ber of metabolism periods. From the results secured in period 
6 it appears that there is a slightly less intense endogenous metab- 
olism as the animal becomes older, as evidenced by a decreased 



THE PROTEINS OF COTTONSEED MEAL 575 

excretion of urinary nitrogen per 100 grams live weight. The 
slight increase in the case of rat 7 during period 6 as compared 
with period 1 is evidently due to a deficient food consumption 
during the former period, necessitating the catabolism of body 
protein to furnish energy. The individual values obtained in 
period 1 for urinary nitrogen per 100 grams live weight are used 
in the subsequent tables for calculating the utilization of the 
various rations, the values always being corrected to the average 
live weight of the particular animal during that period. 

The quantity of fecal nitrogen per 100 grams feed when the 
rat is consuming a nitrogen-free ration is quite a constant factor, 
although this relationship seems to be affected somewhat by ex- 
tremes in the amount of feed consumed, as may be noted in the 
case of rats 4 and 7, period 6. Perhaps a more potent factor in 
causing this fluctuation is the varying amount of filter paper 
eaten, as found by Mitchell (20) in an experiment in this labora- 
tory in which rats during one period had no access to filter paper 
and during the other actually consumed some paper. 

It is recognized that the calculation of the metabolic nitrogen 
in the feces by the method described is subject to an error when 
applied to a variety of rations. Were the content of crude fiber 
in all rations the same as in the synthetic nitrogen-free ration, 
the assumption that the metabolic nitrogen of the feces varies 
directly with the amount of feed consumed would be valid, but 
with rations varying as widely in the percentage of crude fiber 
as the corn and alfalfa rations, the adoption of such an assump- 
tion evidently leads to an error of undetermined magnitude. 
However, the method of correcting the absorbed nitrogen and the 
nitrogen balance, by the use of the factor for metabolic fecal 
nitrogen obtained on nitrogen-free rations, undoubtedly gives 
values nearer the truth than if no such corrections were made, 
since the actual metabolic fecal nitrogen on the experimental 
rations containing protein was very probably greater per 100 
grams of food consumed than the factors used. In computing 
the amount of metabolic fecal nitrogen shown in the tables that 
follow, the values for fecal nitrogen per 100 grams feed eaten in 



576 W. B. NEVENS 

period 1 in the case of each animal are applied to the data for the 
same animals in later periods. 2 

If it is true, as seems probable from the meager data obtained, 
that the endogenous metabolism of the rat becomes less intense 
per unit of live weight as the animal approaches maturity, then 
in conducting investigations of the kind under consideration it 
would, no doubt, be advisable to introduce a metabolism period 
using a nitrogen free ration every six or eight weeks. With these 
data available it would be possible to make linear corrections for 
any changes in the requirement of the basal metabolism. In 
the tables showing the utilization of the proteins which follow, 
such corrections have not been attempted with the limited num- 
ber of data at hand, but should the data secured with rats 1 and 4 
be used for such a purpose, it would necessitate changes in the 
utilization coefficients given of not more than 1 to 2 per cent 
as a maximum. 

Palatability of rations. The results obtained in the six metab- 
olism periods during which nitrogenous rations were fed are 
summarized in table 8. 

From an inspection of the figures giving the amounts of feed 
consumed daily it is evident that all rations containing cotton- 
seed meal were readily consumed by the animals, attesting to 
the palatability of this feed, even when forming as much as 24 
per cent of the ration. When corn was the sole source of protein 
in the ration the amounts of feed consumed were smaller than 
with any other ration. Ground corn seems to be less palatable 
to rats than whole corn for the latter is usually eaten readily. 
Perhaps another reason why less of the corn ration was consumed 
than the alfalfa ration, for example, is that the corn ration was 
much more digestible and had a higher calorific value, so that 
less of it was required to supply the energy requirements. The 
ration in which cottonseed meal and corn were combined was con- 
sumed in greater quantities than the corn ration but not so freely 
as the cottonseed meal ration. Alfalfa hay proved very palatable, 
as all rations of which it formed a part were readily eaten. 

2 These values, as well as those for urinary nitrogen, when being used for these 
calculations, were extended to one more decimal place than shown in table 7. 



TABLE 8 



The utilization of the proteins of cottonseed meal, corn and alfalfa hay; 
summary of results 



BAT 

NUMBER 


PERIODS 


RATION 


M 
> 

3-1 

4 



H 

2 
P 

Z 

O *H 

3 

H a 


go 

O M 

n H 
< 


h. k 

gig 
< 


m 

O H 

z o 

H O 
O K 
O En 
° 5 
H 


°S 

fc 

o m ■ 
c n & 

n O O 
8 aj O 

d 5 « 


a 
ag 

oh 

P5 K 








grams 


grams 


mgm. 


mgm. 


mgm. 


per 
cent 


per 
cent 


1 


2 + 3 


Cottonseed meal 


98 


9.50 


124.8 


54.5 


25.3 


64 


30 


2 


2 + 3 




109 


8.62 


107.1 


38.2 


30.4 


64 


19 


3 


2+3 




127 


11.99 


169.6 


91.3 


28.0 


71 


44 


Average 






111 


10.04 


133.8 


61.3 


27.9 


66 


31 


1 


4 + 5 


Cottonseed meal + 


114 


10.83 


125.9 


52.9 


30.6 


67 


26 


2 


4 + 5 


alfalfa hay 


126 


11.06 


131.7 


58.1 


35.7 


71 


29 


3 


4 + 5 




159 


15.68 


185.0 


89.5 


33.7 


67 


34 


Average 






133 


12.52 


147.5 


66.8 


33.3 


68 


29 


2 


6 + 7 


Cottonseed meal + 


136 


9.82 


124.3 


45.6 


38.2 


63 


21 


3 


6 + 7 


alfalfa hay + corn 


186 


14.01 


173.0 


72.5 


39.4 


65 


27 


Average 






161 


11.92 


148.7 


59.0 


38.8 


64 


24 


4 


2 + 3 


Corn 


118 


7.75 


118.9 


34.6 


22.8 


48 


15 


5 


2 + 3 




108 


6.98 


105.9 


24.7 


27.1 


49 


8 


5 


2 + 3 




115 


6.92 


104.5 


34.1 


20.3 


52 


16 


Average 






114 


7.22 


109.8 


31.1 


23.4 


49 


13 


4 


4 + 5 


Cottonseed meal + 


129 


9.81 


141.4 


59.5 


24.9 


60 


30 


5 


4 + 5 


corn 


118 


8.30 


127.8 


43.6 


29.2 


59 


21 


6 


4 + 5 




124 


8.11 


120.2 


48.9 


22.3 


60 


28 


Average 






124 


8.74 


129.8 


50.7 


25.5 


60 


26 


5 


6 + 7 


Cottonseed meal + 


139 


10.90 


136.9 


49.3 


34.9 


62 


18 


6 


6 + 7 


alfalfa hay + corn 


141 


11.20 


146.8 


64.8 


25.1 


61 


28 


Average 






140 


11.05 


141.9 


57.1 


30.0 


61 


23 


7 


2 + 3 


Alfalfa hay 


104 


9.42 


99.4 


37.2 


21.4 


60 


16 


8 


2 + 3 




103 


8.69 


92.9 


33.7 


20.0 


58 


11 


9 


2 + 3 




121 


12.55 


128.1 


61.2 


29.4 


70 


30 


Average 






109 


10.22 


106.8 


44.0 


23.6 


62 


19 


7 


4 + 5 


Alfalfa hay + corn 


115 


12.64 


149.1 


56.8 


23.3 


54 


20 


8 


4 + 5 




118 


14.08 


175.8 


78.9 


23.3 


59 


28 


9 


4 + 5 




134 


12.51 


166.6 


70.6 


32.7 


62 


29 


Average 






122 


13.08 


163.8 


68.8 


26.4 


58 


26 


8 


6 + 7 


Alfalfa hay + corn 


142 


11.81 


152.6 


65.0 


27.0 


61 


26 


9 


6 + 7 


+ cottonseed meal 


154 


13.06 


184.0 


76.1 


38.0 


62 


29 


Average 






148 


12.83 


168.3 


70.6 


33.0 


61 


27 



577 



578 W. B. NEVENS 

Utilization of proteins. In the summary of results shown in 
table 8 two methods of calculating the utilization of the proteins 
fed are included for comparison, although, for reasons discussed 
above, the second method, namely, the utilization of the absorbed 
nitrogen for both maintenance and growth, is employed in the 
discussion here. 

The utilization of the nitrogen absorbed from a cottonseed 
meal ration containing 10 per cent of crude protein was found to 
be 66 per cent, using the average results of six metabolism 
periods with three rats. The utilization of the proteins of 
alfalfa hay was found to be only slightly less than that of 
cottonseed meal, namely, 62 per cent. 

When these two feeds were combined in such proportion that 
each furnished about an equal amount of digestible protein to 
the ration, very interesting results were secured, indicating a 
slight supplementary effect of the proteins from these two sources. 
This effect was not pronounced, the utilization percentage 
being 2 per cent above that of cottonseed meal alone and 6 per 
cent above that of alfalfa hay alone. It is noted that during the 
periods when the cottonseed meal alfalfa hay ration was fed, 
greater quantities of feed were consumed and larger amounts of 
nitrogen were absorbed than with either the cottonseed or 
alfalfa hay rations alone, which may in some unknown way have 
operated in effecting a more efficient utilization of the nitrogen, 
although the same conditions hold true in the case of both groups 
of rats which received either the corn or the alfalfa ration during 
two periods and were then changed to rations containing proteins 
from both sources. 

It was found that the proteins of corn were utilized the least 
efficiently of those of the three feedingstuffs compared. When 
corn was combined with cottonseed meal or with alfalfa hay the 
resulting utilization coefficients tended toward a mean of the 
utilization coefficients secured with these feedingstuffs when 
fed alone, but were nearer that of the feed other than corn. For 
example, the utilization coefficients found for the corn and 
cottonseed meal rations were 49 per cent and 66 per cent 
respectively, the mean of these two being 57.5 per cent, but 



THE PROTEINS OF COTTONSEED MEAL 579 

the utilization coefficient found for the cottonseed meal-corn 
ration was 60 per cent. Possibly this represents a slight 
supplementary relationship. 

The results obtained with the ration in which cottonseed 
meal, corn and alfalfa hay were combined were remarkably 
uniform. Of the twelve values obtained with rats receiving this 
ration during two metabolism periods each, the lowest value was 
57 per cent and the highest 67 per cent, the average of all being 
63 per cent. The combination of the proteins from three dif- 
ferent sources failed to indicate any farther supplementary effect 
of the proteins. 

The high nutritive value of the proteins of cottonseed meal 
manifested by these experiments is in substantial accord with 
the conclusions of Richardson and Green (1), Osborne and 
Mendel (3, 4, 5, 6) and McCollum and Simmonds (6). They do 
not seem to be in harmony with the findings of Hart and Hum- 
phrey (8) who studied the utilization of the proteins of cottonseed 
meal for milk production, but since growth and milk production 
are dissimilar functions an absolute comparison of the results of 
the two experiments is not valid. 

Correlation of chemical composition with nutritive value. In 
seeking for an explanation of the differences in the nutritive value 
of the proteins of these feedingstuffs based upon differences in 
their chemical makeup, it is evident first of all that their nutri- 
tive values do not vary so widely as the analytical data at hand 
would indicate. For example, the differences found between 
the utilization of the proteins of cottonseed meal and alfalfa 
hay was but 4 per cent, while from an examination of the data 
in table 5 of a preceding paper, it is apparent that cottonseed 
meal contains more than twice as much arginine nitrogen and 
nearly twice as much histidine nitrogen as alfalfa hay, while the 
latter contains more than three times as much nonprotein nitro- 
gen as the former. 

Several theories may be advanced in explanation of this ap- 
parent inconsistency. In the first place, alfalfa hay is shown 
to have a lower digestibility than either cottonseed meal or corn. 
There is no evidence to preclude the possibility that the char- 



580 W. B. NEVENS 

acter of the nitrogen absorbed from alfalfa hay differs qualita- 
tively from that remaining in the undigested residues. Judging 
from the ease with which tyrosine is split off from proteins in 
tryptic digestion in vitro, it is possible that the absorbed nitro- 
gen contains a greater proportion of amino acids essential to the 
body than the unabsorbed portion. Further, the sterochemical 
arrangement of the amino acids in the protein molecule may 
affect the extent to which the digestive enzymes are able to 
cause hydrolysis of the different proteins. A second considera- 
tion is the possible interchangeability of the various forms of 
nitrogen in nutrition, as already pointed out in the case of arginine 
and histidine. To what extent the nonprotein nitrogen of alfalfa 
hay is utilized in maintenance is problematical, but since there is 
no reason to doubt that the degradation products of crude 
protein are able to serve in this capacity, it is possible that a large 
part of the absorbed nonprotein nitrogen fulfills some of the 
requirements of the animal body. 

It is reasonable to assign the higher content of the basic amino 
acids of cottonseed meal as the reason for its superiority over the 
proteins of alfalfa and corn. In the case of the last mentioned 
feedingstuff, there is the additional factor of a comparatively 
low lysine content to be considered, although from the studies 
of Osborne and Mendel concerning the lysine requirements 
for growth, a lysine content of 2.2 per cent of the protein appears 
to be ample for normal growth. 

In the absence of further information respecting the char- 
acter of the mono-amino acid and nonprotein nitrogen content 
of these feedingstuffs, a detailed picture of which the Van Slyke 
analysis does not include, correlations between the chemical 
composition and nutritive value of the proteins of feedingstuffs 
can proceed little beyond the realm of the functions and rela- 
tionships of the basic amino acids. 

Comparison of feed consumption with that of farm animals. It 
was noted during the course of this investigation that the rats 
consumed an enormous amount of feed in proportion to their 
live weights. In some few cases the amount of air dry feed 
eaten daily was equivalent to as much as 10 or 11 per cent of the 



TABLE 9 
Comparison of feed consumption by albino rats with that of farm animals 















FEED 


SPECIES OR BREED 


LENGTH 




NUM- 
BER OF 


AV- 


AV- 


EATEN 
DAILY 


AND 


OF FEEDING 


FEEDS IN RATION 


ANI- 


ERAGE 


ERAGE 


PER 100 


CLASS OP ANIMAL 


PERIOD 




MALS 
FED 


AGE 


WEIGHT 


POUNDS 

LIVE 
WEIGHT 




days 






days 


pounds 


pounds 




28 


Corn + oats + alfalfa 


10 


227 


854 


2.2 


Percheron 




hay 










fillies* 


28 


Corn + oats + alfalfa 
hay 


10 


683 


1484 


1.8 




35 


Corn + linseed meal + 


4 




978 


2.5 


Hereford 

steersf 




clover hay 










28 


Corn + linseed meal + 


4 




1466 


1.5 






clover hay 












30 


Corn + bran + linseed 


4 


365 


472 


2.9 


Jersey heifers£< 


90 


oil meal + alfalfa hay 

Corn + bran + linseed 

oil meal + alfalfa hay 


4 


730 


839 


1.8 




30 


Corn + bran + linseed 


4 


365 


656 


2.4 


Holstein J 




oil meal + alfalfa hay 










heifers % 1 

f 


90 


Corn + bran -f- linseed 
oil meal -f alfalfa hay 


4 
174 


730 


1112 

38 


1.8 
6.0 








495 
105 




128 
320 


3.8 
2.4 


Sheep** { 










45 
127 


2.1 
1.1 




42 


Cottonseed meal -f- corn 
+ alfalfa hay + syn- 


9 




129ft 


8.4§§ 


Albino rats . . .< 




thetic mixture 












21 or 


18 per cent protein 


17 




175 


4.6§§ 




more*** 








200ft 





* From Bui. 192, 111. Agr. Exp. Sta. 

f From Bui. 197, 111. Agr. Exp. Sta. Data concerning "Full-feed lot." 

t From Nebr. Agr. Exp. Sta. Unpublished manuscript. Data concerning 
"Heavy fed groups." 

§ From Henry and Morrison, Feeds and Feeding, 15th ed. p. 569. 

** Calculated from data of Weiske as quoted by Armsby, The Nutrition of 
Farm Animals, p. 432. 

ff Grams. 

§§ Grams per 100 grams live weight. 

*** From Osborne and Mendel. Protein Minima for Maintenance. Jour. 
Biol. Chem., 1915, xxii, 241. 

581 



582 W. B. NEVENS 

live weight. It seemed of interest to compare the feed consump- 
tion of albino rats with that of farm animals. Such a comparison 
is made in table 9. The tabulations of horses and cattle include 
in each case two entries of the same group of animals at dif- 
ferent ages and weights. It is known that the horses and cattle 
were restricted in the amount of concentrates consumed but were 
offered roughage to practically the limit of their appetites. Hence 
a comparison of the feed consumption of these animals with that 
of the first group of rats, which were the ones concerned in this 
investigation, is warranted, but the data are indicative only. 
Data for the amount of feed consumed by the swine, sheep and 
second group of rats is not at hand. 

It is evident from the data presented that the rat is a voracious 
eater, even when receiving rations comparable to those of farm 
animals. A rough approximation places the relative amounts 
of feed eaten by rats as about three times that of various breeds 
and classes of farm animals, if swine be excepted. The fact is 
also brought out, as has previously been noted by others, that 
the young aDimal consumes much more feed in proportion to 
live weight than when older and heavier. 

Cottonseed meal probably not toxic to albino rats. None of the 
rations containing cottonseed meal seemed to exert any harmful 
influence upon the rats consuming it. Three of the animals 
received continuously for 7 weeks rations containing from 7.7 
per cent to 23.9 per cent cottonseed meal with no evidence of 
toxic symptoms but remained in excellent nutritive condition. 
This observation is in agreement with those of Richardson and 
Green (1) and Osborne and Mendel (5). 

SUMMARY OF THE DISCUSSION OF THE NUTRITIVE VALUE 
OF THE PROTEINS 

Evidence is presented to show that metabolism experiments 
with the rat as a subject can be carried out with a high degree 
of accuracy. 

Different methods of expressing the nutritive value of the 
proteins of f eedingstuffs are discussed. The plan of employing the 



THE PROTEINS OF COTTONSEED MEAL 583 

results secured in a preliminary and final metabolism period 
during which the animal receives a nitrogen free ration, for the 
calculation of the percentage of the absorbed nitrogen utilized, 
is favored. In comparing the nutritive value of the combined 
proteins of the f eedingstuffs cottonseed meal, alfalfa hay and corn, 
it was found that when one of these feeds furnished the sole 
source of protein in rations containing 10 per cent of crude pro- 
tein, the utilization of the proteins for the growth of albino rats 
was, in the order in which the feedingstuffs are named, 66 per 
cent, 62 per cent and 49 per cent, respectively. 

When rations containing these feedingstuffs, combined in 
various ways, but with each feed furnishing an equal amount of 
digestible protein, were fed, there was evident no clear cut sup- 
plementary effect of the proteins of one feed upon another, 
except in the case of the combination cottonseed meal and alfalfa 
hay, which showed a slight effect. 

No symptoms of toxicity were noted as a result of feeding 
rations containing cottonseed meal over a period of seven weeks. 

When suitable rations are provided, the albino rat consumes 
an enormous amount of feed * in proportion to its live weight. 

The writer desires to express his appreciation of the assist- 
ance of Dr. H. H. Mitchell in outlining the method used in 
this investigation and for many helpful suggestions. He is also 
indebted to Dr. H. S. Grindley for his encouragement and gen- 
eral supervision of the thesis problem. 



584 



W. B. NEVENS 







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THE PROTEINS OF COTTONSEED MEAL 



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THE JOURNAL OF DAIRY SCIENCE, VOL. IV, NO. 



588 W. E. NEVENS 

REFERENCES 

(1) Richardson, A. E., and Green, H. S..: Jour. Biol. Chem., 1916, xxv, 307; 

ibid., 1917, xxx, 243; ibid., 1917, xxxi, 379. 

(2) Mendel, L. B.: Jour. Amer. Med. Assoc, 1915, lxiv, 1539. 

(3) Osborne, T. B., and Mendel, L. B.: Zeit. physiol. Chem., 1912, lxxx, 307. 

(4) Osborne, T. B., and Mendel, L. B.: Jour. Biol. Chem., 1916, xxvi, 293. 

(5) Osborne, T. B., and Mendel, L. B.: Jour. Biol. Chem., 1917, xxix, 69; 

ibid, 1917, xxix, 289; Proc. Soc. Exp. Biol, and Med., 1915-16, xiii, 147. 

(6) McCollum, E. V., and Simmonds, N.: Jour. Biol. Chem., 1917, xxxii, 347. 

(7) Hart, E. B., and Humphrey, G. C: Jour. Biol. Chem., 1917, xxxi, 445. 

(S) Hart, E. B., and Humphrey, G. C: Jour. Biol. Chem., 1918, xxxv, 367. 
(9) Fraps, G. S.: Bui. 128, Texas Agr. Exp. Sta. 

(10) Henry, W. A., and Morrison, F. B. : Feeds and Feeding. 15th ed., Appen- 

dix table 2. 

(11) Mendel, L. B., and Fine, M. S.: Jour. Biol. Chem., 1911, xi, 1. 

(12) Rather, J. B.: Bui. 163, 1913, Texas Agr. Exp. Sta.; Jour. Amer. Chem. 

Soc, 1914, xxxvi, 584. 

(13) Pomaski, A.: Soobshch. Biuro Chastn. Rast. (Petrograd), 1915, II, no. 2, 

3-12; Exp. Sta. Rec, xxxvi, 805. 

(14) Wells, C. A., and Ewing, P. V.: Bui. 119, 1916, Ga. Agr. Exp. Sta. 

(15) Editorial. Exp. Sta. Rec 1910, xxii, 501. 

(16) Dinwiddie, R. R.: Bui. 76, 1902, Ark. Agr. Exp. Sta. 

(17) Withers, W. A., and Brewster, J. F.: Jour. Biol. Chem., 1913, xv, 161. 

(18) Withers, W. A., and Carruth, F. E.: Jour. Agr. Res., 1915, v, 261; Science, 

1915, xli, 324; Jour. Agr. Res., 1918, xii, 83; Jour. Biol. Chem., 1917, 
xxxii, 245. 

(19) Alsberg, C. L., and Schwartz, E. W.: Jour. Pharm. and Exp, Therap., 

1921, xvii, 344. 

(20) Mitchell, H. H.: The Nutritive Value of the Protein Mixtures of Food- 

stuffs at Different Levels of Intake. Unpublished manuscript. 

(21) Osborne, T. B., and Mendel, L. B.: Jour. Biol. Chem., 1917, xxxii, 309. 

(22) Osborne, T. B., and Wakeman, A. J.: Jour. Biol. Chem., 1919, xl, 383. 

(23) Waters, H. J.: Proc. Soc. Prom. Agr. Sci., 1908, xxix, 3. 

(24) Aron: Biochem. Zeit., 1910, xxx, 207. 

(25) Folin, O.: Amer. Jour. Physiol., 1905, xiii, 66; ibid, 1905, xiii, 117. 

(26) Landergren, E.: Skan. Arch, fur Physiologie, 1903, xiv, 112. 

(27) Cathcart, E. P.: Biochem. Zeit., 1907, vi, 109. 

(28) Thomas, Karl: Arch. Anat. und Physiol., Physiol. Abt., 1919, 219. 

(29) Rubner, M.: Arch. f. Hygiene, 1908, lxvi, 1. 

(30) Michaud, L.: Zeit. physiol. Chem., 1909, lix, 405. 

(31) Cathcart, E. P.: The Physiology of Protein Metabolism. 1912, p. 68. 

(32) Sherman, H. C: Jour. Biol. Chem., 1920, xli, 97. 

(33) Lewis, H. B., Dunn, M. S., and Doisy, E. A.: Jour. Biol. Chem., 191S, 

xxxvi, 9. 

(34) Mitchell, H. H.: Jour. Biol. Chem., 1918, xxxvi, 501. 

(35) Osborne, T. B., and Mendel, L. B.: Jour. Biol. Chem., 1914, xvii, 325. 



BIOGRAPHY 

The writer was born at Winnebago, Illinois, November 27, 
1885. Academic training: graduated from Rockford High School 
in 1905; attended Wheaton College 1905-6 and College of Agricul- 
ture, University of Wisconsin, 1911-14; Graduate student, Univer- 
sity of Illinois, 1914-17 and 1919-21. Degrees: B. S., University of 
Wisconsin, 1914; M. S., University of Illinois, 1917. Positions: 
Assistant in Dairy Husbandry, University of Illinois, 1914-17; 
Assistant Professor of Dairy Husbandry, University of Nebraska, 
1917-19. Honor Societies: Alpha Zeta, Gamma Sigma Delta, Phi 
Lambda Upsilon and Sigma Xi. 



PUBLICATIONS 

1. Raising Dairy Calves.- Ext. Bui. 51, Nebr. Agr. Exp. Sta., Aug., 1918. 

2. Some Factors Affecting the Cost of Milk Production. Ext. Bui. 55, Nebr. 
Agr. Exp. Sta., June, 1919. 

3. The Arrangement of Rectangular Dairy Barns, Cir. 199, 111. Agr. Exp. 
Sta., June, 1917. (Jointly with R. S. Hulce). 

4. Feed and Care of the Dairy Calf. Cir. 202, 111. Agr. Exp. Sta., Aug., 1917, 
(Jointly with R. S. Hulce). 

5. Care and Management of the Dairy Herd. Cir. 204, 111. Agr. Exp. Sta., 
June, 1919. (Jointly with R. S. Hulce). 

6. Purebred Sires Effect Herd Improvement. Cir. 8, Nebr. Agr. Exp. Sta., 
July, 1919. (Jointly with M. N. Lawritson and J. W. Hendrickson). 

7. Dairy Barn and Milk House Arrangement. Cir. 6, Nebr. Agr. Exp. Sta., 
Oct., 1919. (Jointly with J. H. Frandsen). 

8. Breed and Size of Cows as Factors Affecting the Economy of Milk Pro- 
duction. Jour. Dairy Sci. Vol. II, No. 2, Mar., 1919. 

9. The Preparation of a Dairy Exhibit. Journal Dairy Sci., Vol. II, No. 5, 
Sept., 1919. 

10. The Self Feeder for Dairy Calves. Jour. Dairy Sci., Vol. II, No. 6, Nov., 
1919. 

11. The Quantitative Determination of Amino-Acids of Feeds. Jour. Biol. 
Chem. 1921, XLVIII, 249-272. (Jointly with T. S. Hamilton and H. S. Grindley). 



